Method for the production of a dairy food product and a method for gene transfer by conjugation

ABSTRACT

The present invention relates to the field of microbiology, specifically the field of production of a dairy food product using fermentation and the field of gene transfer by conjugation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. patent applicationSer. No. 16/084,591, filed 13 Sep. 2018, which is the National Stageentry of International Application No. PCT/EP2016/055676, filed 16 Mar.2016. The disclosure of the priority applications are incorporated intheir entirety herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 C.F.R. § 1.821-825 (seeM.P.E.P. § 2442.03(a)), a Sequence Listing in the form of anASCII-compliant text file (entitled “Sequence_Listing_2919208-481001_5T25.txt” created on 16 Jun. 2021, and 11,849 bytes in size) is submittedconcurrently with the instant application, and the entire contents ofthe Sequence Listing are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of microbiology, specificallythe field of production of a dairy food product using fermentation andthe field of gene transfer by conjugation.

BACKGROUND OF THE INVENTION

Lactic acid bacteria (LAB) are gram-positive bacteria that are generallyregarded as safe and they are used extensively in food and feedfermentations. They are also found on mucosal surfaces of humans andanimals (34, 37). One of the dominant features during fermentationprocesses with LABs is that they produce lactic acid as the mainmetabolic end product and this leads to rapid acidification and hencepreservation of the fermented material. An additional functionality ofmany strains is the production of volatile metabolites that areimportant flavour compounds (51-53). In fermentation the bacteria canalso play a significant role in altering textural properties of thematerial through e.g. proteolytic activity or the production ofextracellular polysaccharides (50).

In general the structure of fermented dairy products is very complex,consisting of caseins, whey proteins, fat droplets, serum or wheypockets, minerals, salts and microorganisms. Herein, we refer to suchstructure as the matrix. In such a matrix interactions between milkcomponents and their functionality are studied extensively (14, 24, 31,35, 46). For example, the rheological properties of a milk gel depend onthe size and number of fat droplets and the nature of availableemulsifiers (43). If the matrix is stabilized by low molecular weightsurfactants, then the milk gel is weak and has a high meltability, theextent to which gel flows and spreads upon heating, because the surfaceof fat droplets is smooth and non-interactive. However, if fat dropletsare stabilized by whey proteins, then the milk gel is strong and has alow meltability because protein-protein cross-linking interactions areformed between emulsified fat droplets. Other interactions in the foodmatrix can be hydrophobic (30), electrostatic (11), hydrogen bonding(64), Van der Waals, depletion interaction (59), steric repulsion (16)and salt bridges (5).

In contrast to interactions between the milk components themselves verylittle is known about so called microbe-matrix interactions whichdescribe the interaction between lactic acid bacteria and matrixcomponents of the fermented products (7-10, 47).

Interactions between microorganisms and milk components occur viasurface properties of both particles. Bacteria as well as matrixcomponents have a charge and hydrophobicity. The surface properties ofbacteria are determined by molecular composition of its cell wall, whichcan be decorated with (lipo-) teichoic acids, proteins, pili, or(exo/capsular)polysaccharides (EPS/CPS) (13, 22, 45). The molecularcomposition of the cell wall has a significant impact on the roughnessof the bacterial surface, on bacterial chaining and on cell aggregation.These properties govern the interactions between bacteria and the foodmatrix (7, 41). A well-studied example of a change in texturalproperties is the production of EPS by the bacterial culture used forthe fermentation. EPS are hydrocolloids that can bind high amounts ofwater, increasing water holding capacity in protein-free pores of thefermented milk matrix. This leads to an increase in milk viscosity andit reduces syneresis (2, 26, 48). Additionally, the charge, stiffnessand linearity of EPS molecules impact on rheological and physicalproperties of the fermented milk matrix. EPS modification by partialremoval of side groups leads to its reduced efficiency as thickener(58).

Besides the role of EPS on textural properties of fermented milk littleis known about the influence of bacterial surface properties oninteractions with the matrix and its functional consequences on flavourand texture.

SUMMARY OF THE INVENTION

In an aspect, the invention provides for a dairy bacterial strain withclumping and/or chaining properties, preferably when cultured underliquid conditions, preferably without a substantial amount of a clumpingagent, wherein the clumping property results in clumps of at least 20bacteria per average clump and wherein the chaining property results inchains of at least 8 bacteria per average chain.

In a further aspect, the invention provides for a method for theproduction of a dairy bacterial strain according to the invention,comprising:

-   -   culturing a precursor dairy bacterial strain under conditions        that are conducive to the development of clumping and/or        chaining properties and, optionally, isolating a dairy bacterial        strain according to the invention.

In a further aspect, the invention provides for a dairy bacterial strainobtainable by a method according to the invention.

In a further aspect, the invention provides for a composition comprisinga dairy bacterial strain according to the invention.

In a further aspect, the invention provides for a method for theproduction of a food product, preferably a dairy food product, morepreferably a fermented milk product, from a precursor food productcomprising inoculating the precursor food product with a strainaccording to the invention or with a composition according to theinvention and incubating the inoculated precursor food product.

In a further aspect, the invention provides for a food product,preferably a dairy food product, obtainable by the method according tothe invention.

In a further aspect, the invention provides for a food product,preferably a dairy food product, comprising a dairy bacterial strainaccording to the invention.

In a further aspect, the invention provides for the use of a dairybacterial strain according to the invention, or of a compositionaccording to the invention for the preparation of a food product,preferably a dairy food product.

In a further aspect, the invention provides for a bacterial straincomprising an increased amount of expression product of a pilin genecluster, wherein the amount of expression product is increased in viewof Lactococcus lactis subsp cremoris NCDO712.

In a further aspect, the invention provides for a method for genetransfer by conjugation comprising, providing a composition comprisingat least one bacterial strain according to the invention and incubatingthe composition under conditions conducive to conjugation.

In a further aspect, the invention provides for a method for theproduction of a desired bacterial strain comprising a genetic trait ofinterest, comprising providing a composition comprising:

-   -   a precursor of the desired bacterial strain, wherein the        precursor lacks the genetic trait of interest, and    -   a donor bacterial strain comprising the genetic trait of        interest, incubating the composition under conditions conducive        to conjugation, and optionally isolating the desired bacterial        strain comprising the genetic trait of interest, wherein at        least one bacterial strain in the composition comprises a pilin        gene cluster.

In a further aspect, the invention provides for a bacterial straincomprising a genetic trait of interest obtainable by a method accordingto the invention

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found a novel gene cluster that encodes pilinstructures. These pilin structures surprisingly enhance conjugationefficiency and provide texturing properties to the bacteria expressingthe pili gene cluster. Accordingly, in a first aspect the presentinvention provides for a dairy bacterial strain with clumping and/orchaining properties, preferably when cultured under liquid conditions,preferably without substantial amounts of a clumping agent, wherein theclumping property results in clumps of at least 20 bacteria per averageclump and wherein the chaining property results in chains of at least 8bacteria per average chain. Said bacterial strain is herein referred toas a dairy bacterial strain or a bacterial strain according to theinvention; individual bacteria from such strain are herein referred toas bacteria according to the invention.

The term “dairy bacterial strain” is herein defined as any bacterialstrain that is suitable for use in a dairy product and/or in theproduction of a dairy product. Dairy products are known to the personskilled in the art; preferred dairy products are the dairy productsdescribed elsewhere herein. The terms “culturing” and “fermenting” areherein used interchangeably. The term “clumping” is herein defined asthe aggregation under liquid conditions of bacteria according to theinvention into a single aggregate or clump (said terms are usedinterchangeably herein) comprising twenty bacteria or more. Preferably,the average aggregate or clump comprises at least 20, at least 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100bacteria according to the invention. Preferably, the average aggregateor clump comprises between 20 and 100 bacteria according to theinvention, more preferably between 40 and 60 bacteria according to theinvention. The term “chaining” is herein defined as the arrangement of anumber of bacterial cells according to the invention into a chain orchord-like structure. Preferably, the average chain comprises at least8, at least 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, or at least 100 bacteria according to the invention.Preferably, the average chain comprises between 8 and 60 bacteriaaccording to the invention. Preferably, at least 10%, at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the bacteria accordingto the invention are part of a clump or chain. In the invention, theclumping and/or chaining properties of the bacteria according to theinvention are intrinsic properties of a bacterial strain according tothe invention; said properties are present without the induction by anexternal agent. An external clumping agent is ethanol or salts such asammonium sulfate. The term “without substantial amounts of a clumpingagent” means that such agent is not present in an amount that wouldinduce clumping of bacteria according to the invention. Preferably, lessthan 10%, less than 5%, 4%, 3%, 2%, 1%, 0.5%, or most preferably lessthan 0.1% of clumping agent is present. In case of ethanol, preferablyless than 5%, less than 4%, 3%, 2%, 1%, 0.5%, or most preferably lessthan 0.1% (v/v) ethanol is present. In case of ammonium sulfate,preferably less than 2M, less than 1M, 0.5M, 0.4M, 0.3M, 0.2M, or mostpreferably less than 0.1M is present.

Preferably, a dairy bacterial strain according to the invention,comprises an increased amount of expression product of a pilin genecluster. Expression is herein understood to include any step involved inthe production of a polypeptide including, but not limited totranscription, post-transcriptional modification, translation,post-translational modification, and secretion. Accordingly, anexpression product is herein defined as any product within the processof expression, including preRNA, mRNA, pre-polypeptide and maturepolypeptide. A preferred expression product is a mature polypeptide,more preferably a cluster of polypeptides arranged in the form of apilin. The pilin gene cluster is a cluster of genes which finalexpression product is a pilin and may comprise several different genes,such as a sortase gene and one or more genes encoding one or morerespective pilin subunits. The pilin cluster is preferably organizedinto a single operon. Preferably, the amount of expression product ofthe pilin cluster is increased in view of the expression of said pilincluster in Lactococcus lactis subsp cremoris NCDO712 when cultured andassayed under identical conditions. The person skilled in the art knowshow to determine the amount of expression product. Methods fordetermining the amount of expression product (expression level) include,but are not limited to, measurement of (m)RNA using Northern blot or(quantitative) PCR and measurement of protein using Western blot oranother immune-based assay. Also within the scope of the presentinvention is a dairy bacterial strain derived from a parental strainwherein the expression of a pilin cluster is higher in the derivedstrain as compared to the parental strain, such that the clumping and/orchaining properties of the derived strain are higher as compared to theparental strain. Such strain may be derived by recombinant techniques orby classical techniques such as mutagenesis, preferably followed byscreening for a mutant strain comprising the desired properties.

In an embodiment, the dairy bacterial strain according to the inventionis a dairy bacterial strain with clumping and/or chaining propertieswherein the clumping property results in clumps of at least 20 bacteriaper average clump and wherein the chaining property results in chains ofat least 8 bacteria per average chain, and comprising increased amountof expression product of a pilin gene cluster.

In an embodiment, the dairy bacterial strain according to the inventionis a dairy bacterial strain with clumping and/or chaining propertieswherein the clumping property results in clumps of at least 20 bacteriaper average clump and wherein the chaining property results in chains ofat least 8 bacteria per average chain, wherein the amount of expressionproduct is increased in view of Lactococcus lactis subsp cremorisNCDO712 when cultured under identical conditions.

In an embodiment, the dairy bacterial strain according to the inventionis a dairy bacterial strain with clumping and/or chaining properties,wherein the clumping property results in clumps of at least 20 bacteriper average clump and wherein the chaining property results in chains ofat least 8 bacteria per average chain, wherein the pilin gene clusterhas a nucleotide sequence that has at least 30% sequence identity withSEQ ID NO: 1, or a part thereof.

In a preferred dairy bacterial strain according to the invention, thepilin gene cluster has a nucleotide sequence that has at least 30%, atleast 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or most preferably at least 100%sequence identity with SEQ ID NO: 1, or a part thereof.

“Sequence identity” is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (nucleotide, polynucleotide) sequences, as determined bycomparing the sequences. In the art, “identity” also means the degree ofsequence relatedness between amino acid or nucleic acid sequences, asthe case may be, as determined by the match between strings of suchsequences “Identity” can be readily calculated by known methods,including but not limited to those described in (Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heine, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the GCG program package (Devereux etal., 1984). BestFit, BLASTP, BLASTN, and FASTA (Altschul, et al., 1990).The BLAST X program is publicly available from NCBI and other sources(BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894).The well-known Smith Waterman algorithm may also be used to determineidentity.

Preferred parameters for polypeptide sequence comparison include thefollowing: Algorithm: Needleman and Wunsch (1970); Comparison matrix:BLOSSUM62 from Hentikoff and Hentikoff (1992); Gap Penalty: 12; and GapLength Penalty: 4. A program useful with these parameters is publiclyavailable as the “Ogap” program from Genetics Computer Group, located inMadison, Wis. The aforementioned parameters are the default parametersfor amino acid comparisons (along with no penalty for end gaps).Preferred parameters for nucleic acid comparison include the following:Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970);Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap LengthPenalty: 3. Available as the Gap program from Genetics Computer Group,located in Madison, Wis. Given above are the default parameters fornucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp toglu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asnor gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu;Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trpto tyr; Tyr to trp or phe; and, Val to ile or leu. Preferably, sequenceidentity is determined by comparing the whole length of a sequence asidentified herein.

A dairy bacterial strain according to the invention may be any suitabledairy bacterial strain known to the person skilled in the art. Apreferred dairy bacterial strain is a lactic acid bacterial strain.Lactic acid bacteria are known to the person skilled in the art and arepreferably defined as Gram positive, non-spore-forming, anaerobic,catalase negative cocci or rods, forming lactic acid as an end productof their carbohydrate metabolism.

Lactic acid bacteria include, but are not limited to, bacterial strainsselected from the group consisting of the genera of Lactobacillus,Lactococcus, Leuconostoc, Carnobacterium, Streptococcus,Bifidobacterium, Bacteroides, Eubacterium, Clostridium, Fusobacterium,Propionibacterium, Enterococcus, Staphylococcus, Peptostreptococcus, andEscherichia, preferably consisting of Lactobacillus and Bifidobacterium.Preferred species of Lactobacillus, Bifidobacterium, Streptococcus,Leuconostoc and Pediococcus, are Lactobacillus reuteri, L. fermentum, L.acidophilus, L. crispatus, L. gasseri, L. johnsonii, L. plantarum, L.paracasei, L. murinus, L. jensenii, L. salivarius, L. minutis, L.brevis, L. gallinarum, L. amylovorus, Streptococcus thermophilus,Leuconostoc mesenteroides, Pediococcus damnosus, P. acidilactici, P.parvulus, Bifidobacterium bifidum, B. longum, B. infantis, B. breve, B.adolescente, B. animalis, B. gallinarum, B. magnum, and B. thermophilum.A more preferred dairy bacterial strain according to the invention is aLactococcus strain, more preferably a Lactococcus lactis strain, evenmore preferably a Lactococcus lactis subsp. lactis strain or aLactococcus lactis subsp. cremoris strain.

The dairy bacterial strain according to the invention with its specificclumping and/or chaining properties when cultured under liquidconditions preferably has the capacity to texturize milk, such as butnot limited to, the capacity to increase viscosity when cultured in milkand capacity to increase stress to shearing when cultured in milk. Afurther preferred property of the dairy bacterial strain according tothe invention is that it influences the bacterial localization incheese. A dairy bacterial strain according to the invention has theproperty that an increased fraction of bacteria remain in curd duringcheese making Fifty percent to 80% of bacterial strains that are not thebacterial strain according to the invention typically remains in thecurd. With a the dairy bacterial strain according to the invention, thepercentage bacteria in the curd can be increased to at least 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%. Accordingly, thedairy bacterial strain according to the invention preferably has thecapacity to increase the fraction of bacteria that remain in curd duringcheese making. The cheese can be any cheese and preferably is a cheeseas defined herein below. The percentage bacteria that remain in the curdis preferably increased to at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or at least 99%. Preferably, the fraction of bacteria thatremain in the curd is increased by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or to at least 100% as compared to the fraction ofbacteria that remain in the curd when using a bacterial strain that isnot a bacterial strain according to the invention. The person skilled inthe art knows how dairy bacterial strains can texturize milk, an exampleis the thickening of milk when producing yoghurt, thus increasingviscosity. A further example is the gel strength, also called gelhardness of fermented milk, such as of yoghurt and cheese. Preferably,the texturizing capacity of a dairy bacterial strain according to theinvention is higher than the texturizing capacity of Lactococcus lactissubsp cremoris NCDO712 when cultured and assayed under identicalconditions. When the dairy bacterial strain according to the inventionis a dairy bacterial strain derived from a parental strain such asdefined previously herein, the texturizing capacity of said derivedstrain is preferably compared to the texturizing capacity of theparental strain; the texturizing capacity of the derived bacterialstrain preferably being higher than the texturizing capacity of theparental bacterial strain. Higher texturizing capacity preferably is atleast 10%, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 400%, 500%, or at least 1000% higher respective texturizingcapacity.

It is understood that replicates of a diary bacterial strain accordingto the invention are encompassed by the invention. The term “replicate”refers to the biological material that represents a substantiallyunmodified copy of the material, such as material produced by growth ofmicro-organisms, e.g. growth of bacteria in a culture medium.

In a further aspect, the invention provides for a method for theproduction of a dairy bacterial strain according to the invention,comprising:

-   -   culturing a precursor dairy bacterial strain under conditions        that are conducive to the development of clumping and/or        chaining properties and, optionally, screening for a dairy        bacterial strain according to the invention and/or isolating a        dairy bacterial strain according to the invention. Preferably,        the clumping and/or chaining properties of the dairy bacterial        strain obtained by the method are kept for at least 5, at least        10, 15, 20, 25, 30, 35, 40, 45, or at least 50 generations,        allowing e.g. the dairy bacterial strain with the clumping and        chaining properties to be used as a starter strain for the        production of a dairy food product. Preferably, the clumping        and/or chaining properties of the dairy bacterial strain        obtained by the method are (also) kept during storage such as        cold storage (between 0 and 4 degrees Celsius), storage at room        temperature and longer term storage in freeze dried, frozen or        liquid suspension state. The conditions to be applied to provide        the clumping and/or chaining properties to the dairy bacterial        strain may be any condition available to the person skilled in        the art. Preferably, such condition comprises at least one of        mutating the dairy bacterial strain, preferably by chemical        treatment, radiation treatment and/or recombinant treatment.        Such method is preferably followed by screening for and/or        isolating a dairy bacterial strain according to the invention.        The person skilled in the art knows classical mutagenesis        methods using e.g. chemical treatment and/or radiation        treatment. As an example such method comprises providing a dairy        bacterial strain, subjecting such strain to mutagenesis by        chemical treatment and/or radiation treatment and screening for        a dairy bacterial strain according to the invention. When the        method is a recombinant method, it preferably comprises bringing        a pilin gene cluster, or a part thereof, to overexpression in a        dairy bacterial strain, so as to provide the clumping and/or        chaining properties according to the invention said dairy        bacterial strain. Methods for overexpressing a gene cluster, or        a part thereof, are known to the person skilled in the art. The        invention further provides for a dairy bacterial strain        obtainable by the method of this aspect of the invention.

In a further aspect, the invention provides for a composition comprisinga dairy bacterial strain according to the invention. Such compositionmay or may not be a kit of parts. Such composition is herein referred toas a composition according to the invention. Such composition may be anyuseful composition, it may e.g. be a food product, food-grade substanceor a formulation and may be in any form or state of constitution knownto the person skilled in the art. Preferably, in a composition accordingto the invention, the concentration of bacteria according to theinvention ranges from about 1 E−8 weight percent 1 to about 1 weightpercent, more preferably from about 5 E−8 to about 0.9 weight percent,more preferably from about 5 E−8 to about 0.8, more preferably fromabout 5E−8 to about 0.7, more preferably from about 5 E−8 to about 0.6,more preferably from about 1 E−7, 2 E−7, 3 E−7 or 4 E−7 to about 5 E−7weight percent with respect to the total weight of the formulation,and/or about 1 E+4, 1 E+5, or 1 E+6 to about 1 E+8, 1 E+9, or about 1E+10 colony forming units/ml of composition, even more preferably about1 E+6 to about 1 E+10 colony forming units/ml of composition.

More preferably, in a composition according to the invention, theconcentration of bacteria according to the invention ranges from 1 E−8weight percent 1 to 1 weight percent, more preferably from 5 E−8 to 0.9weight percent, more preferably from 5 E−8 to 0.8, more preferably from5E−8 to 0.7, more preferably from 5 E−8 to 0.6, more preferably from 1E−7, 2 E−7, 3 E−7 or 4 E−7 to 5 E−7 weight percent with respect to thetotal weight of the formulation, and/or 1 E+4, 1 E+5, or 1 E+6 to 1 E+8,1 E+9, or 1 E+10 colony forming units/ml of composition, even morepreferably 1 E+6 to 1 E+10 colony forming units/ml of composition. Acolony forming unit is a term known to the person skilled in the art andone unit typically refers to the amount of bacteria that forms onecolony on a culture plate; it is a term to refer to viable bacteria.Even more preferably, in a composition according to the invention, theconcentration of bacteria according to the invention ranges from 1 E−7to 5 E−7 weight percent with respect to the total weight of thecomposition and/or 1 E+6 to 1 E+10 colony forming units/ml ofcomposition. A composition according to the invention may furthercomprise another dairy bacterial strain. Such further dairy bacterialstrain may be present in the same, single, composition or may be presentseparately in a kit of parts. Such further dairy bacterial strain may beany dairy bacterial strain defined elsewhere herein. A preferred furtherdairy bacterial strain is a Streptococcus thermophilus and/or aLactobacillus species. Another preferred further bacterial strain may bea probiotic bacterial strain. Probiotic bacteria are known to the personskilled in the art and preferably are bacteria of a Bifidobacteriumspecies or lactic acid bacteria e.g. of a Lactobacillus rhamnosusspecies. Within the scope of the invention is the composition accordingto the invention as a starter culture for the production of a foodproduct, preferably in freeze dried, frozen or liquid suspension state.A composition according to the invention comprising a dairy bacterialstrain according to the invention, may also itself be a food product. Afood product as defined herein is a substance suitable for consumptionby a subject, preferably a human or an animal, more preferably a human.A food product may be of plant or animal origin, and may containessential nutrients, such as carbohydrates, fats, proteins, vitamins,and/or minerals. A food product may be intended for ingestion by anorganism and subsequently assimilation by the organism's cells toproduce energy, maintain life, and/or stimulate growth. A food productaccording to the invention includes but is not limited to a substanceselected from the group consisting of a dairy-, grain-, vegetable-,fruit-, fish-, or meat-based product; a preferred food product accordingto the invention is dairy food product, preferably a yoghurt, a curd ora cheese. A preferred cheese is selected from the group consisting ofGouda, Cheddar, Edam, Brie, Camembert, Stilton, Gorgonzola, Blue cheese,Goat cheese, Swiss cheese such as Emmental, Gruyere Brick, Maasdam, andMozarella. The term “based product” is herein defined that the foodproduct is produced from a specific raw material or precursor foodproduct such as dairy, grain, vegetable, fruit, fish, or meat. The foodproduct may be based on a mixture of different raw materials e.g. amixture of dairy and grain including legumes, a mixture of dairy andfruit or a mixture of meat and fruit.

In a further aspect, the invention further provides for a method for theproduction of a food product according to the invention, preferably adairy food product according to the invention, more preferably afermented milk product such as a yoghurt or a cheese, from a precursorfood product comprising inoculating the precursor food product with adairy bacterial strain according to the invention or with a compositionaccording to the invention and incubating the inoculated precursor foodproduct. When the food product according to the invention is a cheese ora yoghurt, the precursor food product is preferably milk, preferably amilk selected from the group consisting of cows, buffalo, goats, sheepsor mixes thereof. Depending on the food product to be produced theperson skilled in the art knows to select to proper temperature and timefor the incubation. In the method according to the invention for theproduction of a food product, preferably a further ingredient is addedto the incubation, preferably an ingredient selected from the groupconsisting of a flavoring agent, a fruit or fruit concentrate, a syrup,a prebiotic, a bacterial strain, such as a probiotic strain, a coloringagent, a thickening agent, a preserving agent, a sweetener, and anenzyme. A thickening agent is preferably a starch, a starch derivative,a cellulose derivative, a gelatin, gum Arabic, a carrageenan, gellangum, xanthan gum, guar gum, and locust bean gum. A thickening agent mayalso be an enzyme such as a transglutaminase. A sweetener may be anysweetener known to the person skilled in the art, such as a sugar, apolyol sweetener, Stevia or an intense sweetener, or mixtures thereof.The polyol sweetener is preferably a sweetener selected from the groupconsisting of sorbitol and xylitol. The intense sweetener is preferablya sweetener selected from the group consisting of aspartame,acesulfame-K and a saccharin. The invention further provides for a foodproduct, preferably a dairy food product according to the invention,obtainable by a method according to this aspect of the invention. Theinvention further provides for a food product, preferably a dairy foodproduct, comprising a dairy bacterial strain according to the invention.When the food product is a cheese, the cheese preferably has anincreased fraction of bacteria that remain in the curd, as defined hereabove, compared to the fraction of bacteria that remain in the curd whenusing a bacterial strain that is not a bacterial strain according to theinvention. Preferably, the food product, preferably a dairy food productaccording to the invention is packaged in a suitable container. Thecontainer is preferably a closed container which is capable to preventor retard oxygen gas from diffusing from the atmosphere into the foodproduct. Preferably, said container further comprises a headspace abovethe food product, wherein preferably more than 90% (v/v) occupied by theheadspace is provided as an inert gas such as nitrogen or carbondioxide. Accordingly, preservation of the food product is enhancedand/or survival of the dairy bacterial strain according to the inventionis enhanced.

The invention further provides for the use of a dairy bacterial strainaccording to the invention, or of a composition according to theinvention for the preparation of a food product, preferably a dairy foodproduct.

The inventors have demonstrated that a dairy bacterial strain accordingto the invention comprises increased conjugation efficiency. Thisproperty is not limited to dairy use of the bacterial strain.Accordingly, in a further aspect, the invention provides for a bacterialstrain comprising an increased amount of expression product of a pilingene cluster, wherein the amount of expression product is increased inview of Lactococcus lactis subsp cremoris NCDO712. Such strain is hereinreferred to as a bacterial strain according to the invention or abacterial strain according to the invention with increased conjugationproperties, or a bacterial strain according to the invention withincreased conjugation efficiency; it should be noted that this aspect ofthe invention is not limited to dairy bacterial strains but applies toany bacterial strain that has conjugation capacity, at least to someextent. Preferably, the conjugation efficiency is increased with atleast 10%, at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200% (two-fold), 500% (five-fold), ten-fold, 20-fold, 30-fold,40-fold, 50-fold, sixty-fold, seventy-fold, eighty-fold, ninety-fold,100,-fold, 1000-fold, 10,000-fold, 100,000-fold or at least1000,000-fold in view of Lactococcus lactis subsp cremoris NCDO712. Whena bacterial strain with increased conjugation efficiency is derived froma parental strain, the conjugation efficiency and the above depictedincrease thereof may be compared to the parental strain where the strainaccording to the invention is derived from. In a preferred bacterialstrain according to the invention, the pilin gene cluster has anucleotide sequence that has at least 30% percentage sequence identitywith SEQ ID NO: 1, or a part thereof. More preferably, the pilincluster, has at least 35%, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or mostpreferably at least 100% sequence identity with SEQ ID NO: 1 or a partthereof.

The invention further provides for a method for gene transfer byconjugation comprising, providing a composition, referred to ascomposition for gene transfer, comprising at least one bacterial strainaccording to the invention and incubating the composition underconditions conducive to conjugation. The person skilled in the art isaware of gene transfer by conjugation and knows how to perform thetechnique. Preferably, the composition for gene transfer comprises atleast two bacterial strains, one bacterial strain being a bacterialstrain according to the invention.

The invention further provides for a method for the production of adesired bacterial strain comprising a genetic trait of interest,comprising providing a composition comprising:

-   -   a precursor of the desired bacterial strain, wherein the        precursor lacks the genetic trait of interest, and    -   a donor bacterial strain comprising the genetic trait of        interest,

incubating the composition under conditions conducive to conjugation,and optionally isolating the desired bacterial strain comprising thegenetic trait of interest, wherein at least one bacterial strain in thecomposition is a bacterial strain according to the invention.

The invention further provides for a bacterial strain comprising agenetic trait of interest, obtainable by a method according to thisaspect of the invention.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

The word “about” or “approximately” when used in association with anumerical value (e.g. about 10) preferably means that the value may bethe given value (of 10) more or less 0.1% of the value.

The sequence information as provided herein should not be so narrowlyconstrued as to require inclusion of erroneously identified bases. Theskilled person is capable of identifying such erroneously identifiedbases and knows how to correct for such errors. In case of sequenceerrors, the sequence of the polypeptides obtainable by expression of thegenes as represented by SEQ ID NO: 2 should prevail.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

Sequences

SEQ ID NO Description Sequence 1 Pilin gene See sequence listing cluster2 Leader See sequence listing sequence 3 pLP712H_FWGGACCAGATGGTACTTTTGAAGCG 4 pLP712H_RV GGTAAAGTCACTATTGATGGACAGCC 5pilinPstI CCGctgcagTTTGCAACAGAACCGTA forw ATTGATTAGC 6 pilinXhoICGGctcgagTTAAGTAATTTGAATTA rev CTTGCTTTGAGAGTTGTTTAAAGG

FIGURE LEGENDS

FIG. 1. Plasmid maps of pSH73, pSH74, and pNZ712. Plasmid pNZ712includes genes encoding functional nisin immunity (nisCIP) and copperresistance (lcoRSABC). A 16-kb plasmid pSH74 contains a novel 8-kb pilusgene cluster spaCB-spaA-srtC1-srtC2. Plasmid pSH73 harbors repX, repBand cadCA genes. The latter encode a cadmium resistance regulatoryprotein and a cadmium efflux ATPase.

FIG. 2. Pilin gene cluster and their suggested functions. The pilincluster consists of spaCB-spaA-srtC1-srtC2 genes with 2 insertionregions IS1216 at the ends. The pilin cluster contains two adjacent srtCgenes encoding sortase C proteins, which probably would catalyze theassembly of pilin proteins into pili. The spaA gene encodes SpaA proteinand we suggest that SpaA is the major pilin backbone subunit. The spaCand spaB genes are fused, but SpaCB is expected to be cleaved by sortaseC. SpaB is expected to be the basal pilus subunit and linked to thepeptidoglycan by sortase A. SpaC has an “E box” (YALTETKTP) and avonWillebrand type-A domain (vWFA). The “E box” is necessary to linkSpaC to SpaA proteins. It has been speculated that the SpaC segmentmight be involved in bacterial adhesion to surfaces and might be thepilin tip protein.

FIG. 3. Cell aggregation after pilus over-expression. A—L. lactisNCDO712, B—empty vector control MG1363(pIL253), C—MG1363(pIL253pilΔI),D—MG1363(pIL253pil). The images were taken in 3 minutes afterre-suspending the cells in the buffer, pH 6.8.

FIG. 4. Pilin expression in L. lactis leads to increased chain length.A—MG1363pIL253, B—MG1363(pIL253pil), C—MG1363(pIL253pilΔI).

FIG. 5. Scanning electron microscopy of pilin overexpressing L. lactisstrains. A—NCDO712, B—IL1403, C—IL1403(pIL253pil), D—MG1363(pIL253),E—MG1363(pIL253pil), F—MG1363(pIL253pilΔI). White errors indicate piliin panel C. White bars: 2 μm in all panels. All panels have the same 50000× magnification.

FIG. 6. Scanning electron microscopy of pilin overexpressing L. lactisstrains. A—NCDO712(pIL253pil), B—NCDO712, C—MG1363(pIL253pil),D—MG1363(pIL253). White bars: 1 μm in all panels.

FIG. 7. Microstructure of milk fermented by L. lactis strains withaltered surface properties. Bacterial cells are

, while proteins

and fat droplets

form aggregated matrix; the black areas represent the serum fraction.The black bar in the right corner displays the 25 (B) L. lactis MG1363expressing pili shown chaining, clumping, hydrophobic phenotype seemedto be located in serum regions compared to (A) its control strain, (C)clumping and hydrophobic transconjugant MG1614 harboring pLP712 seem tobe attached to protein matrix, (D) chaining MG1363 seems to go throughthe protein matrix and serum regions, (E) pili expressing MG1299 showncell chaining, clumping and hydrophobicity similar to (D) does not showexact pattern of localization in milk matrix, (F) EPS producing MG1363seems prefer localization in serum regions.

FIGS. 8A-8D. Model: bacteria as structure elements in fermented foods.

is fat droplet (Ø=1 μm, can be up to 5-6 μm, 10¹⁰ droplets/ml)stabilized by whey proteins (Ø=4-6 nm, 10⁷⁷ proteins/ml),

—casein micelle (Ø=200 nm, 10¹⁴ micelles/ml),

—bacterial cell (Ø=1 μm, 10⁹ cell/ml). FIG. 8A shows the structure ofmilk fermented with unmodified cell surface: the fat droplets and cellsare part of casein network with cavities in which the serum phase isconfined. Such a gel has a weak network. FIG. 8B shows the structure ofmilk fermented with pili over-expressing cells that locate in cavitieswhere aqueous phase should be located (consistent with chaining andclumping lactococcal phenotypes studied here). FIG. 8C shows thestructure of milk fermented with chaining cells. The gel structure isweak because of spaces in the casein network and long bacterial chainswhich probably work as structure breakers; but the viscosity of suchfermented milk increases possibly due to long cell chains which increasesolid content. FIG. 8D shows the gel structure of milk with cavities inprotein matrix fermented with EPS producing strains.

FIG. 9.

Types of interactions in milk between bacterial cell and milkcomponents. The covalent bond (e.g. C-C) possesses 350 kJ/mol, van derWaal's—10 kJ/mol, electrostatic—15 kJ/mol, and hydrogen—21 kJ/mol. Forcebetween micellar casein (or denatured whey) protein and cell for LGG isabout 0.4 nN (7), and it is very strain-dependent.

EXAMPLES

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

Unless stated otherwise, the practice of the invention will employstandard conventional methods of molecular biology, virology,microbiology or biochemistry. Such techniques are described in Sambrooket al. (1989) Molecular Cloning, A Laboratory Manual (2^(nd) edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; inSambrook and Russell (2001) Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and2 of Ausubel et al. (1994) Current Protocols in Molecular Biology,Current Protocols, USA; and in Volumes I and II of Brown (1998)Molecular Biology LabFax, Second Edition, Academic Press (UK);Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization(Hames and Higgins, eds.).

Example 1. Plasmid Complement of Lactococcus lactis NCDO712 Reveals aNovel Pilin Gene Cluster

Introduction

Lactococcus lactis is a Gram-positive, non-pathogenic, non-spore forminglactic acid bacterium (LAB) that is often isolated from plant materialor a dairy environment (75, 159). It is widely used in the dairyindustry as a starter culture for the production of cheese, butter milkand quark. Strains of L. lactis typically contain one to eight differentplasmids (67, 93) ranging from 1 kb (69) to more than 100 kb in size(110, 146). The plasmids often specify traits of industrial importancesuch as growth on lactose, milk protein utilization and stressresistance (68, 83). Examples are the 5 plasmids of L. lactis SK11, aphage-resistant dairy strain used in cheese making (143), and in the 7plasmids of L. lactis IL594, the parent of the plasmid-free strainIL1403 (100). Other important plasmid-encoded functions for L. lactisinclude bacteriocin production (74, 81, 85, 92, 104) and resistance (77,101, 120), antibiotic resistance (92, 136) and bacteriophage resistance(66, 84, 130, 131, 148, 149). Also metal ion resistance systems arefound frequently to be plasmid encoded (129). Moreover, a recentpublication described a CRISPR-Cas system that was encoded on alactococcal plasmid, although it was concluded not to be functional(125). In addition several genes related to lactococcal surfaceproperties are carried on plasmids (109, 125, 132), such as aggL—a generesponsible for cell auto-aggregation, or genes responsible for adhesionto mucus (105). One of the most intensively studied L. lactis strains isMG1363, a plasmid-cured derivative of strain NCDO712 (95, 118). NCDO712was originally isolated from a dairy starter culture and was shown toharbour 5 plasmids with molecular sizes of 33, 9, 5.2, 2.5, and 1.8 MDa(95). During plasmid curing of strain NCDO712, derivatives harboringindividual plasmids were obtained, allowing a targeted analysis ofplasmid-encoded functions (95). Important biotechnological properties ofthe strain, namely lactose utilization and proteolysis, were linked tothe 33 MDa (55 kb) plasmid pLP712 (95, 160). This lactose/proteaseplasmid pLP712 (160) can be transferred to other lactococcal strains byconjugation (96). Conjugation can occur through various rearrangementswhich include co-integrate formation between pLP712 and a genome-encodedsex factor (SF). The co-integrate is roughly double the size of pLP712since the SF is 59.498 kb (97). After conjugation approximately half ofthe transconjugants displayed an aggregating phenotype and transferredthe lactose/protease plasmid with high-frequency (71, 96, 107, 117,158). The aggregating phenotype is only seen in transconjugants carryingthe pLP712-SF co-integrate (97, 148) and it was linked to the cluA genewhich is located on the SF (99, 148). The cluA gene encodes a surfaceprotein involved in cell-to-cell contact and cell aggregation (99, 148).

Some L. lactis strains also express proteinaceous surface appendagescalled pili (113, 123, 130). Pili are known to have different functionsin bacteria, including adhesion to surfaces (type I pili) or motility(type IV pili) (94, 121, 113, 122). One of the best described is the sexpilus involved in conjugation in Escherichia coli (80). Pilinbiosynthesis genes can be encoded by the chromosome (132) or on plasmids(125) and they are described to be involved in cell aggregation (155),bacterial adherence to host cells (105, 151, 157) or attachment toenvironmental substrates/surfaces (150). Two plasmids of L. lactisNCDO712 have already been sequenced and published. Plasmid pLP712 (55395bp) harbors the genes for lactose import and catabolism, theextracellular protease PrtP, and genes encoding extracellular proteins,transposases, and hypothetical proteins (160). Plasmid pSH71, thesmallest one (2062 bp) (88), is highly similar to pWV01 (88, 116) andboth of them are the basis of a broad range of lactococcal cloningvectors (88, 111, 112, 126, 127, 138).

We here sequenced the plasmids of NCDO712 and found that contrary toearlier reports it contains 6 but not 5 plasmids (95). The additionalplasmid encodes functional nisin immunity and copper resistance genesand on one of the other plasmids we could identify a novel pilin genecluster which we showed to be functional by overexpression analysisfollowed by phenotypic characterizations.

Materials and Methods

Bacterial Strains, Growth Conditions and Medium.

L. lactis subsp. cremoris NCDO712 (95) and its derivatives (Table 1)were grown at 30° C. in M17 (Oxoid, Thermo Scientific, Hampshire, UK)supplemented with 1% glucose (GM17). When required, erythromycin (Ery;10 μg/ml), chloramphenicol (Cm; 5 ug/ml), rifampicin (Rif; 50 μg/ml),and streptomycin (Str; 100 μg/ml) were added to the indicated finalconcentrations. The lactose-positive L. lactis strains NDO712, SH4109,and MG1299 were grown in M17 containing 1% lactose (LM17). Allincubations were carried out at 30° C.

TABLE 1 Strains and plasmids used in this study. Strain or plasmidCharacteristics Reference L. lactis strains NCDO712 L. lactis dairyisolate (pLP712, pSH71, pSH72, (95) pSH73, pSH74, pNZ712) This studyMG1363 Plasmid-cured derivative of L. lactis NCDO712 (95) SH4109Prophage-cured derivative of L. lactis NCDO712 (95) containing all 6plasmids found in this study MG1388 A phage T712 lysogen derived from L.lactis (95) MG1363 MG1362 Derivative of L. lactis NCDO712 (95) (harborspSH72) MG1063 Derivative of L. lactis NCDO712 (95) (harbors pSH73 andpSH72) MG1261 Derivative of L. lactis NCDO712 (95) (harbors pSH73)MG1365 Derivative of L. lactis NCDO712 (95) (harbors pSH71) MG1299Derivative of L. lactis NCDO712 (95) (harbors pLP712) NZ9700 Nis^(R);Derivative of L. lactis MG1363; (72) pepN::nisRK MG1614 Str^(R) andRif^(R) derivative of L. lactis MG1363 (95) IL1403 Plasmid-freederivative of L. lactis IL594 (75) Plasmids pIL253 Ery^(R); 4.9 kb; Lowcopy-number derivative of (145) pAMβ1 pIL253pil Ery^(R); 13.1 kb; pIL253harboring pSH74 pilin This study operon spaCB-spaA-srtC1-srtC2 with 300bp upstream region pIL253pilΔ1 Ery^(R); 11.6 kb; pIL253 harboringspaCB-spaA- This study srtC1-srtC2 with 1,5-kb internal deletion inspaCB

Sequencing of L. lactis NCDO712 Total DNA and Sequence Assembly.

Total DNA of L. lactis NCDO712 and derivatives was isolated usingphenol-chloroform extraction as previously described (140) with thefollowing modifications. The exponentially growing cells were harvestedby centrifugation (10 min at 6240 g) after which the cell pellet wasre-suspended in THMS buffer (30 mM Tris-HCL (pH 8), 3 mM magnesiumchloride, 25% sucrose) containing lysozyme (2 mg/ml) and 50 μg/ml RNaseand incubated for 1 h at 37° C. Subsequently, the cells were treatedwith SDS (final concentration 1%) for 20 min at 65° C. After thatproteinase K (0.3 mg/mi) was added and incubation was continued for 10min at 37° C. Total DNA was extracted from the lysate using severalextractions with phenol/chloroform after which it was precipitated withisopropanol. The DNA was dissolved in sterile water.

The purified total DNA was sheared to fragments of approximately 500 bpusing the Covaris ultrasone device (KBioscience, LGC, Köln, Germany) Thepaired-end NEB NExtGen library preparation kit (New England Biolabs,Inc., MA, US) was used according to the manufacturer's instructions toprepare the NGS library. The libraries were 101 bases paired-endsequenced on an Illumina HiSeq2000 (Illumina, Inc., San Diego, Calif.,USA). Velvet (162, 163) was used in combination with VelvetOptimiser(http://bioinformatics.net.au/software.velvetoptimiser.shtml) to performde novo paired-end assembly of the genome. All contigs that did not mapto the L. lactis MG1363 genome were assumed to be plasmid fragments;these were first scaffolded by mapping onto known L. lactis plasmids inthe NCBI database. Further scaffolding was supported by PacBiosequencing (BaseClear, Leiden, the Netherlands) on a 5-kb library ofNCDO712 total DNA. Remaining gaps in the plasmid sequences were closedwith dedicated PCR reactions followed by amplicon sequencing (BaseClear,Leiden, the Netherlands).

Initial automatic annotation of the plasmids was performed using theRAST annotation server (72). Manual curation of plasmid-encoded featureswas performed with Artemis (88, 137), followed by family, domain, motifand context analyses of encoded proteins using BlastP (NCBI) andInterpro (http://www.ebi.ac.uk/interpro/). IS elements and transposasegenes were identified using IS Finder (https://www-is.biotoul.fr//). TheDNA sequences of the assembled plasmids were used for a BLAST search(http://blast.ncbi.nlm nih.gov/Blast.cgi) in the NCBI plasmid databasecontaining complete plasmids. The determination of single nucleotidepolymorphisms of NCDO712 in comparison to the sequenced derivativeMG1363 was performed using the Breseq software package (83) and theGenBank file: NC_009004 in combination with corresponding nextgeneration sequencing data: SRA064225 as templates.

For non-synonymous SNPs the software SIFT (113) and the UniProt-TrEMBLdatabase (http://www.uniprot.org/) were used to predict whether an aminoacid substitution would affect protein function.

Determination of Nisin and Copper Resistance.

Overnight cultures of L. lactis were diluted in fresh M17 medium to afinal optical density at 600 nm (OD₆₀₀) of 0.03. To measure nisinresistance, the medium was supplemented with nisin from L. lactis(N6764-5G, Sigma-Aldrich, Steinheim, Germany) at different endconcentrations (0-20 ng/ml). L. lactis NZ9700 was used as a control(Table 1). The strains were grown in 10 ml sterile tubes for 7 h at 30°C. The OD₆₀₀ was measured after 4 h and after 7 h using a UV/VisibleUltrospec 2000 spectrophotometer (Pharmacia Biotech, Cambridge,England).

To measure copper resistance, CuSO₄ (0-4.8 mM end concentrations) wasadded to the growth medium. A 96 well microplate with the samples wasincubated for 21 h at 30° C. The OD₆₀₀ was measured every 15 min with aSpectraMax spectrophotometer (Molecular Devices, Wokingham, Berkshire,UK).

Pilin Overexpressing in L. lactis.

The spaCBA-srtC1-srtC2 locus (designated as pil locus) including its 300bp upstream region was amplified with KOD Hot Start Polymerase (MerckMillipore, Madison, Wis., USA) using the pilinPstI forward primer (SEQID NO: 5) and the pilinXhoI reverse primer (SEQ ID NO: 6). The purifiedPCR product was digested with PstI and XhoI and ligated to similardigested pIL253 using T4 DNA Ligase (Invitrogen, Breda, TheNetherlands). The ligation mixture was transformed (161) toelectro-competent (102) MG1363 cells. Transformants harbouring theanticipated pIL253pil plasmid (Table 1) were selected using colony PCRconfirmation with primers pLP712H_FW and pLP712H_RV (SEQ ID NO:'s 3 and4, respectively).

An internal deletion of 1451 bp in the spaCB gene was constructed bydigestion of pIL253pil with AatII followed by re-ligation andintroduction of the plasmid in L. lactis MG1363. The resulting plasmidwas designated pIL253pilΔI (Table 1).

Cell Aggregation.

L. lactis cells from a 10 ml overnight culture were washed twice with 10ml sterile 10 mM phosphate buffer, pH 6.8, re-suspended in the samebuffer, after which cell sedimentation was observed visually, while cellchaining was examined by light microscopy.

Scanning Electron Microscopy (SEM). Bacterial cells were cultured for 1day on GM17 agar plates. From plates with 50-100 colonies small piecesof agar gel carrying less than 5 colonies were cut out and placed in amicroscope sample holder. All further steps of cell fixation, washing,dehydration, staining, freeze-drying, electron microscopy, and imageanalysis were performed according to (103). For imaging a FEI Magellan400 FESEM electron microscopy (Wageningen Electron Microscopy Centre,The Netherlands) was used.

Conjugation Experiments.

Conjugation experiment were performed as described previously (124, 147)with L. lactis MG1614 as a recipient strain. Transconjugants wereselected on milk agar plates containing 0,004% bromocresol purple(Merck, Darmstadt, Germany), streptomycin (100 μg/ml) and rifampicin (50μg/ml) when donor strains were NCDO712 or MG1299(pIL253pil).Transconjugants were selected on glucose Elliker agar platessupplemented with streptomycin (100 μg/ml) and erythromycin (10 μg/ml)when donor strain was MG1363(pIL253pil). For the conjugation of pNZ712encoding copper resistance genes from NCDO712 to MG1614 LM17 plates weresupplemented with streptomycin (100 μg/ml), rifampicin (50 μg/ml) and1.2 mM CuSO₄.

Results

Chromosomal Differences Between L. lactis NCDO712 and MG1363.

Re-sequencing of the total DNA of NCDO712 allowed the detection of 11Single Nucleotide Polymorphisms (SNPs) between the chromosomes of L.lactis NCDO712 and its plasmid-cured derivative MG1363 (159), which wasisolated in 1983 following multiple rounds of chemical- andprotoplast-induced plasmid curing (95). Amongst the 11 SNPs found in thechromosome of L. lactis NCDO712, three are synonymous, three are inintergenic regions, while the other five lead to amino acid changes inproteins. The sequencing data also suggests the occurrence of genomere-arrangements but their verification was out of scope of this study.

Only one of the three SNPs in the intergenic regions is predicted to bein a promoter region, that of the mtlA gene encoding a putativemannitol-specific PTS system EIIBC component. For mtlA the differentialRNA sequencing has pinpointed the transcription start site (TSS) atposition 26465 (van der Meulen et al. accepted). The mutation at theposition 26455 suggests that the −10 box is altered from an optimalTATAAT into TACAAT. Furthermore, three of the protein sequence-affectingSNPs in the genes encoding a hypothetical protein and 2 transposaseswere predicted not to affect protein function. These predictions weremade using SIFT, an algorithm that analyzes the effect of mutationsbased on the degree of conservation of amino acid residues (113, 144).Mutation in the gapB and tsf genes encoding the glyceraldehyde3-phosphate dehydrogenase and elongation factor TS respectively arepredicted to affect the protein function. Whether these mutations arecaused by genetic drift or if they confer a fitness advantage in alaboratory environment is unclear. However, the data indicate that thenumber of SNPs occurring between MG1363 and NCDO712 is limited.

L. lactis NCDO712 Harbors Six Plasmids.

Assembly of all nucleotide sequence reads that did not map onto thechromosome of L. lactis MG1363 revealed that L. lactis NCDO712 carries atotal of 6 rather than the previously described 5 plasmids (95). UsingL. lactis NCDO712 derivatives harboring single plasmid species (95) welinked the plasmids identified here to the earlier described plasmids ofthis strain (Table 2). The plasmid sizes did not fully correspond to therespective sizes determined here, which this is most likely caused bythe limitations of size estimation based upon agarose gelelectrophoresis (95). However, we cannot exclude that plasmidrearrangements accounting for (part of) the differences have occurredduring strain propagation over the years (160). The additional plasmididentified in this study, designated pNZ712, has a size similar to thatof pLP712, which may explain why it escaped detection in 1983 (95).

The copy number of the individual plasmids varied between 2 and 4, basedon mean coverage number of chromosomal DNA and plasmid coverage (Table).The plasmid replication mode was determined using previously describedcriteria (68, 119). Rolling circle replication (RCR) was identified onbasis of the presence of Rep-family protein encoding genes and adouble-stranded origin (dso) of replication; while a replicationinitiator protein encoding repB gene and an origin of replication (ori)are indicative of theta-type plasmid replication (68). These analysesindicated that pSH71 replicates through a rolling circle mechanism (68,88), while the other 5 plasmids replicate using a theta-type mechanism(68).

Plasmids pLP712 and pSH71 have been sequenced and described earlier (88,160). The nucleotide sequence of the pLP712 is identical to the onedetermined here, except for a single nucleotide difference, whereas thepSH71 sequence differs by 6 nucleotides (Table) and plasmid origin.

To investigate the relationships of plasmids pSH72, pSH73, pNZ712, andpSH74 with other known plasmids we compared them with 1.955 plasmidsequences in the NCBI database (database consulted on Feb. 1, 2015).pSH72 (3.597 bp) had the highest copy number, approximately 4 copies percell, and only appeared to encode the replication genes repB, repX, andrep C. Except for 3 nucleotide differences pSH72 was identical toplasmid pND324 which was isolated from L. lactis subsp. lactis LL57-1(90) (Table). The biological function of this plasmid is unclear. pSH73is identical to pAG6, a plasmid isolated from Lactococcus lactis ssp.cremoris 712 (accession number: AB198069, GI: 70067197), which is mostlikely the same strain as NCDO712 or a derivative of it (97). The onlySNP (A→G, pAG6→pSH73) detected is at nucleotide position 1143 of thehsdS gene encoding a type I restriction/modification system specificitysubunit. Plasmid pSH73 harbours next to the replication genes repX andrepB also cadCA genes that were predicted to encode a cadmium resistanceregulatory protein and a cadmium efflux ATPase.

The two other plasmids pSH74 and pNZ712 were found to have partialsimilarity to known L. lactis plasmids with 93-99% nucleotide identity.Identities were found in genes encoding for several functions such asreplication, transposases, resolvases, copper resistance associatedgenes and nisCIP of which nisi encodes nisin immunity. Detailed sequenceanalysis of pSH74 identified putative pilin biosynthesis genes which weannotated as spaCB-spaA-srtC1-srtC2.

TABLE 2 The comparison of NCDO712 plasmids with the earlier work.Plasmids annotated in this work Plasmid copy Plasmids described in (95)number*/mean Replication Plasmid Size^($) Plasmid Size coverage^(†) modepLP712 33 MDa, pLP712 55 395 bp 2 (423) Theta ~50 kb pSH71 1.8 MDa,pSH71^(RS) 2 062 bp 3 (673) RCR ~3 kb pSH72^(¥) 2.5 MDa, pSH72 3 597 bp4 (921) Theta ~4 kb (99% identical to pND324) pSH73 5.2 MDa, pSH73 8 663bp 3 (674) Theta ~8 kb (identical to pAG6) pSH74 9 MDa, pSH74 15 518 bp3 (697) Theta ~14 kb — — pNZ712 49 832 bp 2 (471) Theta *Estimated onthe bases sequence coverage in comparison to that of the chromosomalDNA. ^($)Plasmid size in original publication is given in MDa. Plasmidsize in kb is estimated through the relationship-1MDa ds-DNA = 1.52 kb(https://tools.thermofisher.com). ^(†)Coverage number is based on theanalysis of 6 million sequence reads. Chromosomal DNA coverage in thesame analysis was 198. ^(¥)pSH72 differs by 3 bp from pND324 (90) (NCBIreference sequence: NC_008436.1): T1295G, G1384A and C3349-deleted.^(RS)pSH71^(RS) sequence differs from pSH71 (NCBI accession numberA09339; de Vos W.M., 1987) by 6 bp: T712-deleted, T713-deleted,A731-deleted, G803A, -deleted1234T, C1414-deleted.

Nisin Immunity and Copper Resistance are Specified by pNZ712.

Nisin is a lanthionine-containing antimicrobial peptide that binds tolipid II, disrupts the cytoplasmic membrane and causes death ofsusceptible bacterial cells. The nisin operon nisABTCIPRKFEG waspreviously described to be present on the sucrose-nisin transposonTn5276 and carries, next to the nisin structural gene nisA, genesresponsible for modification, transport and precursor cleavage, genesinvolved in the regulation of the nisin operon and genes specifyingresistance to the bacteriocin (126, 156). Plasmid pNZ712 carries nisCIP,but nisC is only partially present. The nisC gene encodes an enzymeinvolved in posttranslational modification (in concerted activity withNisB) of the nisin precursor, nisi encodes nisin immunity, and nisPencodes the serine protease which is involved in maturation of the nisinprecursor.

To determine if nisi is functional, L. lactis NCDO712 was grown in LM17medium supplemented with 0 and 20 μg/ml nisin. L. lactis NZ9700 was usedas a nisin resistant control. In the absence of nisin all strainsreached a maximal OD₆₀₀ of 2.96±0.63 after 7 h of growth. When thestrains were grown in the presence of 20 μg/ml nisin, the positivecontrol NZ9700 reached an OD₆₀₀ of 1.8±0.06. L. lactis NCDO712 andSH4109, strains carrying all 6 plasmids including pNZ712 with theplasmid-encoded nisCIP, reached an OD₆₀₀ of 0.63±0.01 and 0.67±0.09,respectively. The optical density reached is less compared to NZ9700which has the full immunity function. This difference is a lack ofnisFEG, genes encoding an ABC transporter that contributes to nisinimmunity in NCDO712. The plasmid-free derivative L. lactis MG1363 andother derivatives carrying single plasmids but not pNZ712 from L. lactisNCDO712 reached an OD₆₀₀ of only 0.06±0.046. Hence, the pNZ712-encodednisi is functional and provides nisin immunity to L. lactis NCDO712.

To determine if the putative copper resistance genes lcoRSABC on pNZ712are functional, L. lactis NCDO712 and several of its derivatives notcarrying pNZ712 were grown in the presence or absence of CuSO₄. MG1363showed no growth if 0.8 mM or more CuSO₄ was added to the medium. L.lactis NCDO712, harboring pNZ712 with the lcoRSABC genes, was the onlytested strain able to grow in the presence of 1.2 mM CuSO₄, indicatingthat the gene cluster on pNZ712 is functional. At 2, 4 and 4.8 mM ofCuSO₄ NCDO712 started to grow after 5, 10 and 15 hours respectively. Wecould also show that pNZ712 harboring the copper- and nisin-resistancegenes can be transferred from one lactococcal strain to another viaconjugation when copper is used as a selective marker, whichcorroborates the functionality of the genes.

A Novel Pilin Gene Cluster is Present on pSH74.

Plasmid pSH74 was found to harbour an 8-kb gene cluster which weannotated as spaCB-spaA-srtC1-srtC2. It is flanked by IS1216 elements(FIG. 2). Different gene-orders of pilin biosynthesis genes have beenreported in gram-positive bacteria (121, 154), but the gene-order foundon pSH74 mostly resembles that of the spaC-spaB-spaA-srtC cluster ofLactobacillus rhamnosus GG (105, 134, 157) although the encoded proteinsdisplay only 30 to 45% amino acid sequence identity. In addition, theplasmid-encoded pilin cluster of pSH74 contains two adjacent srtC genes,which encode sortase C proteins of 413 and 392 amino acid residues,respectively. The two sortases are only 38% identical, and two or eventhree consecutive srtC genes have previously also been found in thepilin biosynthesis gene clusters in Streptococcus agalactiae, S.pneumoniae, and Clostridium diphteriae (89, 142). Similarly toLactobacillus rhamnosus GG (89, 111, 112), srtC in L. lactis NCDO712might be responsible for the assembly of pilin proteins into pili, whilethe chromosomally encoded srtA (llmg_1449) should recognize LPxTGE orLPxTGD motifs and covalently anchor pili to peptidoglycan of the cellwall (89).

Also similar to L. rhamnosus GG, the SpaA protein of L. lactis NCDO712is presumably the pilin main backbone subunit. It contains the typicalLPSTGGAG motif near the C-terminus for cleavage by sortase C and has thecharacteristic YPKN “pilin motif” with a conserved lysine residue. SpaAalso carries the YVLNETKAP “E box”, which is suggested to have astructural role in pilus assembly (80, 127, 144).

The spaC and spaB genes, which are separate genes in Lactobacillusrhamnosus GG, are fused in L. lactis NCDO712. In L. rhamnosus GG SpaB isthe basal pilus subunit and SpaC is the pilin tip protein. In L. lactisNCDO712 the first approximately 840 amino acid residues of SpaCBcorrespond to SpaC, the remaining C-terminal 260 residues to SpaB. TheSpaC segment in SpaCB contains an LPSTGGAG motif that could be cleavedby sortase C, possibly splitting the SpaCB into 2 separate proteins SpaCand SpaB. Moreover, predicted with PePPER (87) an alternativetranscription start site downstream of the LPSTGGAG motif of the SpaCencoding region of the spaCB gene may support independent synthesis ofonly SpaB. The SpaB resembling part of SpaCB contains a C-terminalLPDTGE motif that is predicted to be targeted by sortase A and serves asa peptidoglycan anchoring sequence (89, 134). RNAseq obtained underdifferent stress conditions revealed that srtC1 and srtC2 are usuallyco-expressed, while there is no correlation in expression levels betweenthe other genes of this gene cluster (van der Meulen et al. accepted).

SpaC has an “E box” (YALTETKTP) and a von Willebrand type-A domain(vWFA). The SpaC segment also contains a collagen-binding domain and twocollagen-binding surface-protein-Cna B-type domains, which might beinvolved in bacterial adhesion to surfaces (IPR008970; SSF49478)(http://supfam.org). Taken together, these structure analyses suggestthat analogous to the L. rhamnosus GG pilin, SpaB forms the basal pilussubunit and SpaC fulfills the tip protein function. We found based onprotein comparisons similar pilus clusters are also present in L. lactissubsp. cremoris CNCM I-1631 contig_071 (accession number: AGHX01000000;LLCRE1631_01806, LLCRE1631_01807, LLCRE1631_01808, LLCRE1631_01809) (97%identity), in L. lactis subsp. lactis 1AA59 contig_056 (accessionnumber: AZQT01000047.1 and AZQT01000000) (100% identity), as well as inthe Leuconostoc citreum genome (accession numbers: WP_048699698,WP_048699696, WP_048699695, WP_048699693) (>90% identity). However, thesequences of those organisms are annotated as hypothetical proteins. Inall three of those strains the spaCB genes are fused as well whichsuggests that this may be of relevance for e.g. the regulation of theprotein's ratio to each other.

The L. lactis NCDO712 Pilin Operon is Functional.

To examine whether the identified putative pilin genes are functional inL. lactis, the entire operon with its native promoter was cloned in themedium-copy number plasmid pIL253 (105), resulting in pIL253pil. Initialattempts to clone the pilin operon downstream of the nisin-induciblenisA promoter failed, and the few clones obtained carried the sameinternal deletion in the spaCB genes. Deletion of a similar internalfragment of 1.5 kb from the spaCB gene in pIL253pil resulted inpIL253pilΔI (Table 1). This deletion in spaCB leads to an out of theframe mutation shortening the SpaCB protein by 591 amino acid residuesand retaining a 394-residue truncated protein. The native pilin operonas well as the one with the spaCB deletion were introduced in L. lactisstrains MG1363 and IL1403. An obvious consequence of the plasmid-basedpilin over-expression was cell aggregation and sedimentation of theculture in both strains (results for MG1363 are shown in FIG. 3). Thecells also grew in chains much longer than 10 cells per chain (FIG. 4).Cells expressing the truncated version of SpaCB displayed neither cellaggregation (FIG. 3) nor cell chaining (FIG. 4).

Scanning electron microscopy (SEM) revealed that cells of L. lactisNCDO712 (FIG. 5, panel A) have a relatively rough surface compared tothose of L. lactis IL1403 (FIG. 5, panel B), which were very smooth. Thecontrol strain L. lactis MG1363(pIL253) showed an intermediate state ofsurface roughness (FIG. 5, panel D). Furthermore, even though L. lactisNCDO712 harbors the pil operon, no pili were visible on the surface ofthese bacteria. The pili were seen in L. lactis NCDO712 only when thepIL253pil plasmid was introduced (data not shown). This indicates thatthe expression level of the pilin operon from the native plasmid, whichwas estimated to be present in 3 copies per cell, is not sufficient forthe detection of pili on the cell surface. However, expression of thesame operon from the plasmid pIL253, which is present in higher copynumbers (145) is sufficient to detect the pili. The introduction ofpIL253pil into L. lactis IL1403 led to pilin-like structures, albeit atrelatively low abundance (FIG. 5, panel C), whereas L. lactis MG1363harbouring pIL253pil clearly carries more appendices on its cell surface(FIG. 5, panel E). In addition, an increased roughness of the cellsurface was observed in both IL1403 and MG1363 when they harbourpIL253pil. Interestingly, MG1363(pIL253pilΔI) cells expressing thetruncated version of spaCB (FIG. 5, panel F) were also decorated withpilus-like structures on the surface, but these appendices appeared tobe more disoriented than those present on MG1363 harbouring pIL253pil(FIG. 5, panels E and F). The observed disorientation could imply thatthe pili of MG1363 harbouring pIL253pilΔI are not appropriately attachedto the peptidoglycan, which would be consistent with the prediction thatthe truncated spaCB lacks the predicted SrtA motif for peptidoglycananchoring. These results revealed that expression of thespaCB-spaA-srtC1-srtC2 pilin gene cluster derived from pSH74 of L.lactis NCDO712 leads to the formation of pilin-like appendices on thesurface of lactococcal cells.

In gram-negative bacteria the length of pili is approximately 1-2 μm,and the diameter is between 1 nm and 10 nm. In gram-positive bacteriapili have been reported to have a length of 0.3-3 μm, and a diameter of3-10 nm (151). In L. rhamnosus GG the length of pili is up to 1 μm andthe diameter is 1-10 nm (105). Pili in L. lactis IL1403 reached up to 3μm in length and a diameter of approximately 5 nm (132). On the surfaceof L. lactis TIL448 pili were short and thin with a length up to 450 nm,but on the surface of the overexpressing strain pili became longer withaverage length of 2 μm and diameter of 2.3 nm (125). Interestingly, thepili overexpressed in MG1363(pIL253pil) from pSH74 of L. lactis NCDO712are shorter and thicker than most others with a length of 200-240 nm anda 18-20 nm diameter, whereas pIL253pilΔ1 derived pili inMG1363(pIL253pilΔI) were thinner with a diameter of approximately 14 nm.

Conjugation Efficiency.

One of the known functions of pili in gram negative bacteria isinvolvement in conjugation (80). To examine whether the L. lactisNCDO712 pil operon might play a role in DNA transfer by conjugation, wemeasured the efficiency of conjugation in the presence and absence ofthe pil operon. L. lactis NCDO712 carries a sex factor in its chromosomethat is involved in co-integrate formation with and subsequent conjugaltransfer of the lactose/protease plasmid pLP712 (97, 99). This processcan be readily quantified using the plasmid-free, lactose deficient L.lactis MG1614 (Lac⁻, PrtP⁻, Strep^(R), Rif^(R)) (Table 1) as a recipientand selecting for lactose-fermenting colonies that are also resistant torifampicin and streptomycin. The efficiency of transfer of pLP712 fromL. lactis NCDO712, MG1299 and MG1363 was up to 20-, 17- and 50-foldincreased upon pili overexpression, respectively (Table 3). We did notfind co-transformation of pIL253pil during the conjugation of pLP712.Although the observed increase in conjugation efficiency is not veryhigh, these observations indicated that pili might contribute to theefficacy of exchange of DNA between lactococcal cells. We do not know ifthis effect is caused by pili mediated cell clumping or through pilithat seem to make cell to cell contact (FIG. 6).

TABLE 3 Results of conjugation experiments. In all cases the strain L.lactis MG1614 was used as the recipient. The strains with pIL253pilshowed an increase in the number of transconjugants obtained per donorCFU. When strain with empty plasmid (pIL253) was tested in conjugationexperiment, pili overexpression enhanced conjugation efficiency by 50fold. Transconjugants per donor CFU Fold Donor Average SD (n = 4)increase NCDO712 1.1E−07 3.54E−08 NCDO712(pIL253pil) 2.4E−06 1.13E−0721.91 MG1299 1.4E−07 4.74E−07 MG1299(pIL253pil) 1.6E−06 1.11E−06 11.18MG1363(pIL253) 8.3E−10 MG1363(pIL253pil) 4.4E−08 53.01

Discussion

Sequencing of the total DNA of L. lactis NCDO712 revealed the presence 6plasmids in this strain of which some specify potentially importantindustrial traits. Functional nisin immunity and copper resistance genesare present on pNZ712 (51.7 kb), a plasmid that was not identified in anearlier study of the plasmids of L. lactis NCDO712 (95). This newlyidentified plasmid possibly escaped detection because pNZ712 has a sizesimilar to that of the lactose/protease plasmid pLP712. Plasmid-encodedcopper resistance has been described previously in Streptococcus (nowLactococcus) lactis (70, 91, 119), but has not been described in L.lactis NCDO712. According to earlier studies lcoRS are regulatory genesinvolved in transcription of lcoABC, while lcoABC itself confers copperresistance by lowering the accumulation of copper inside the lactococcalcell (143). However, next to the lcoRSABC cluster pNZ712 also containspNZ712_23, a protein which is annotated as copper-(orsilver)-translocating P-type ATPase that could also be involved in theincreased copper resistance found NCDO712. Interestingly, in Lactococcuslactis C2, a recently diverged derivative of NCDO712 (86, 114), whichwas reported to carry 5 plasmids, the resistance to metal ions,including copper, was suggested to be encoded on a DNA region of thelactose plasmid pLM3001 of which the sequence remains unknown (91). Ourresults show that copper resistance and lactose utilization genes resideon different plasmids, indicating that plasmid re-arrangements may haveoccurred in NCDO712, C2, or in both strains (86).

We found that pSH74 (17 kb) contains an 8-kb pilus gene cluster. Pilusgenes have been described earlier in L. lactis. Oxaran and co-workers(132) identified a chromosomally encoded pilus gene cluster in L. lactisIL1403, which was initially based on sequence analyses and homologysearches to characterized pilus and sortase proteins. Similar to ourwork with NCDO712, detectable production of pili could only be achievedby overexpression of the pilus genes yhgD, yhgE, yhhA, yhhB in the samestrain. For overexpression they used the medium-copy number vectorpIL253 and the constitutive promoter P23, rather than its nativepromoter as we have used here. The native promoter region of spaCBseemed important for successful expression/cloning of the pilus genecluster, since expression without leader resulted in deletions in spaCBafter expression from other a nisin inducible and the constitutive purCpromoter. The strategy with the native promoter was followed afterobserving a leader sequence of ˜200-nt upstream of spaCB by visualizingRNA-seq data by Rockhopper (123) (van der Meulen, accepted). Based onnegative staining and TEM analysis Oxaran et. al. detected pili understandard laboratory conditions in other L. lactis isolates. These werevegetable isolates such as KF282 and NCDO2118, as well as clinicalisolates such as 2885-86 and 810-85. Interestingly, the chromosomallyencoded pilus gene cluster yhgD, yhgE, yhhA, yhhB of IL1403 appeared tobe present in all lactococcal genomes known to date, including those ofL. lactis strains SK11, KF147, and MG1363 (llmg_1800-1803) (132).

In another study L. lactis TIL448 was shown to carry a plasmid-encoded6.9-kb pilus gene cluster, yhgE2-srtC2-yhhB2-ORF4, that lead to theformation of pili at the cell surface (125) when overexpressed in eitherL. lactis MG1363 or in the wild type strain TIL448. Within this clusteryhgE2 was predicted to encode the pilus backbone, while srtC2 wasproposed to facilitate pilin polymerization. ORF4, the putative pilustip protein, contained a lectin-like domain (PF00139) predicted to havecarbohydrate-binding properties, that were proposed to allow bacterialbinding to plant cell walls. In addition, expression ofyhgE2-srtC2-yhhB2-ORF4 was shown to increase the attachment of L. lactisTIL448 to intestinal epithelial Caco-2 cells (132).

For the pilus cluster identified in NCDO712 we could neither findhomologies at the protein level nor in the organization of the pilusgene clusters when compared to those of L. lactis IL1403 or TIL448.Besides the differences at sequence level and operon organization, thepilus length and diameter seems to differ from most earlier identifiedlactococcal pili (125, 132, 151).

Comparing to well described pilus clusters the gene organization inNCDO712 is closest to that of the pilus operon of L. rhamnosus LGG (86,146). The GC content in the L. lactis NCDO712 pilus operon (35.3%) tothat of the rest of the genome (35.7%) suggests that the acquisition ofthe pilus gene cluster is not a recent event or has been transferredfrom a microbial species with a similar GC content. The latterexplanation may be supported by the presence of a highly homologouspilus cluster in the genome of Leuconostoc citreum (>90% proteinidentity) with a GC content of 38.9%.

Based on gene homology and organization we hypothesize that the functionof the pilin genes of L. lactis NCDO712 is similar to those in Lb.rhamnosus GG (86, 146) and other Gram-positive bacteria (108, 122, 133,144). Pilus formation and attachment to peptidoglycan in Lactobacillusrhamnosus GG is governed by the pilin-specific sortase SrtC1 and thehouse-keeping sortase SrtA. The pilin-specific sortase C specificallytargets a triple glycine motif LPxTGGxG at N-terminal end of pilinprotein, and catalyzes the assembly of pilin proteins into pili (89,121). The chromosomally encoded enzyme sortase A targets N-terminalLPxTGE or LPxTGD motifs (89) and catalyzes the covalent anchoring ofextracellular proteins, including pilin proteins, to peptidoglycan inthe cell wall. The sortase A recognition sequence does not necessarilyprevent its recognition by a sortase other than SrtA (89). It means thatsortases A and C may each be able to recognize both LPxTGD/E andLPxTGGxG motifs used for the regulation of pilin biosynthesispolymerization and anchoring (89), but the protein-structural featuresinvolved in motif recognition by sortases still remain unclear. Forexample, when spaA-srtC1 of Lb. rhamnosus GG were co-expressed in L.lactis NZ9000, it was observed that SrtC1 can recognize and polymerizethe SpaA protein as well as pilus tip protein SpaC, while SrtA is onlyinvolved in anchoring of SpaB to the peptidoglycan. Lb. rhamnosus GGalso carries a pilus gene cluster, spaFED, on its chromosome, where SpaDis the pilus backbone protein and SpaE is located at the base of thepilus structure, like SpaB (135, 157). SpaF, similar to SpaC, is theminor pilin subunit that locates to the tip of the pilus and was shownto be responsible for adhesion of pili-carrying cells to the intestinalmucus (137). L. lactis NCDO712 SpaA and SpaC contain the LPxTGGxG motifin its C-terminus for cleavage by the pilin-specific sortase C forfurther formation of isopeptide bonds to interconnect these pilinsubunits. SpaA also carries the “E box” and YPKN “pilin motif”. The “Ebox” is suggested to have a structural role in pilus assembly (80, 127,144) as is illustrated by the involvement of the “E box” of SpaA fromCorynebacterium diphtheria in the attachment of SpaB to polymerized SpaAfibres (87). The “pilin motif” is involved in intermolecular peptidebond formation between the carbonyl-group carbon of the threonineresidue of the pilin subunit and the side-chain of the lysine in pilinmotif of the neighboring pilin subunit (151). These bonds lead to theformation of membrane-associated covalently-linked dimer with a pilinmotif that can interact with other pilin subunits, forming an elongatedpilus fibre (151). Thereby, the presence of an E-box, LPxTGGxG and YPKNmotifs in L. lactis SpaA suggests that this protein constitutes themajor pilin backbone subunit.

As we described earlier the SpaC segment of SpaCB contains an LPxTGGxGSrtC motif we hypothesize that after cleavage of SpaCB by SrtC, the “Ebox” would link SpaC to SpaA proteins. It has been speculated that thevWFA domain, which is also present in SpaC of L. rhamnosus GG (105), mayhave lectin-like binding properties and could bind to sugars with highcarbohydrate specificity. The SpaB segment in SpaCB carries thecanonical LPxTG motif for cleavage and linkage to peptidoglycan bysortase A. Hence, SpaB is most likely at the base of the lactococcalpilus, and SpaC is the pilin tip protein. An internal deletion in spaCBled to the formation of much more, albeit aberrant and somehowdisoriented, pili at the surface of the cells, which would be inagreement with a lack of the proper attachment of the pili via the basalsubunit SpaB.

Since the Lactobacillus rhamnosus GG pilus operon is involved inattachment to human intestinal epithelial cells, Caco-2, we tested ifthe pilus operon identified here confers similar binding properties.However, no significant differences in attachment to Caco-2 or HT29 (ahuman colonic carcinoma cell line) cells were observed betweenstationary-phase cells of L. lactis NCDO712 or L. lactis either with orwithout pIL253pil (data not shown). The level of adherence oflactococcal cells was relatively low as compared to the level ofadherence displayed by the Lb. rhamnosus GG cells that were used as acontrol in these experiments.

Cell aggregation or close-proximity is an important factor forconjugative plasmid transfer. Conjugative plasmid transfer ingram-positive cocci was studied (79, 101, 141), including lactococci(75, 141, 147), but there was no report on the role of pili in thisprocess. Since pili overexpression leads to increased cell-clumping andwe also observed direct cell-cell contact via pili in SEM images, weinvestigated the impact of pili expression on plasmid transferfrequencies and conclude that pili expression can enhance frequency oftransconjugation by a factor of 11- to 53-fold per donor cell CPU. Theunderlying mechanisms remain unknown, but the pilin tip protein SpaCcould bind to another cell, keeping the two cells in close proximity(80). Such close proximity of cells could facilitate and increase theconjugation frequency. We also cannot exclude that pili could enableconjugation of genetic material to other species.

Example 2. Bacteria as Structural Elements in a Fermented Food Product;Alteration of Lactococcal Surface Properties and its FunctionalConsequences on Fermented Milk

Introduction

Lactic acid bacteria (LAB) are gram-positive bacteria that are generallyregarded as safe and they are used extensively in food and feedfermentations. They are also found on mucosal surfaces of humans andanimal (34, 57). One of the dominant features during fermentationprocesses with LABs is that they produce lactic acid as the mainmetabolic end product and this leads to rapid acidification and hencepreservation of the fermented material. An additional functionality ofmany strains is the production of volatile metabolites that areimportant flavour compound (51, 53). In fermentation the bacteria canalso play an significant role in altering textural properties of thematerial through e.g. proteolytic activity or the production ofextracellular polysaccharides (50). In general the structure offermented dairy products is very complex, consisting of caseins, wheyproteins, fat droplets, serum or whey pockets, minerals, salts andmicroorganisms. Throughout this paper we refer to this structure as thematrix. In such a matrix interactions between milk components and theirfunctionality are studied extensively (14, 24, 31, 35, 46). For example,the rheological properties of a milk gel depend on the size and numberof fat droplets and the nature of available emulsifiers (43). If thematrix is stabilized by low molecular weight surfactants, then the milkgel is weak and has a high meltability, the extent to which gel flowsand spreads upon heating, because the surface of fat droplets is smoothand non-interactive. However, if fat droplets are stabilized by wheyproteins, then the milk gel is strong and has a low meltability becauseprotein-protein cross-linking interactions are formed between emulsifiedfat droplets. Other interactions in the food matrix can be hydrophobic(6), electrostatic (11), hydrogen bonding (64), Van der Waals, depletioninteraction (59), steric repulsion (16) and salt bridges (5).

In contrast to interactions between the milk components themselves verylittle is known about so called microbe-matrix interactions whichdescribe the interaction between lactic acid bacteria and matrixcomponents of the fermented products (7-10, 47).

Interactions between microorganisms and milk components occur viasurface properties of both particles. Bacteria as well as matrixcomponents have a charge and hydrophobicity. The surface properties ofbacteria are determined by molecular composition of its cell wall, whichcan be decorated with (lipo-) teichoic acids, proteins, pili, or(exo/capsular)polysaccharides (EPS/CPS) (13, 22, 45). The molecularcomposition of the cell wall has a significant impact on the roughnessof the bacterial surface, on bacterial chaining and on cell aggregation(see example 1). These properties govern the interactions betweenbacteria and the food matrix (7, 41). A well-studied example of a changein textural properties is the production of EPS by the bacterial cultureused for the fermentation. EPS are hydrocolloids that can bind highamounts of water and therefore, increase milk ropiness and water holdingcapacity in protein-free pores of the fermented milk matrix. This leadsto an increase in milk viscosity and it reduces syneresis (2, 26, 48).Additionally, the charge, stiffness and linearity of EPS moleculesimpact on rheological and physical properties of the fermented milkmatrix. EPS modification by partial removal of side groups leads to itsreduced efficiency as thickener (58).

Besides the role of EPS on textural properties of fermented milk littleis known about the influence of bacterial surface properties oninteractions with the matrix and its functional consequences on flavourand texture.

We here studied how the alteration of bacterial surface propertiesaffect textural parameters and flavour volatiles of fermented milk. Thiswas achieved by using 25 isogenic L. lactis strains which only differedin known surface properties and cell morphologies. We have found thatparticular surface alterations not only lead to distinct differences ingel hardness and viscosity of fermented milk but also to significantalterations in some flavour volatiles. Therefore, based on our resultswe conclude that bacteria can be used as structure elements in fermentedfoods.

Materials and Methods

Bacterial Strains and Growth Conditions L. lactis was grown in M17 broth(Oxoid Ltd, Basingstroke, Hampshire, UK) supplemented wither with 1%glucose (GM17) or with 1% lactose (LM17) at 30° C. When required,erythromycin (Ery; 10 μg/ml), chloramphenicol (Cm; 5 μg/ml), rifampicin(Rif; 50 μg/ml), or streptomycin (Str; 100 μg/ml) were added to theindicated end-concentrations.

TABLE 4 Strains and plasmids used in this study. No L. lactis strainsCharacteristic Reference 1 NCDO712 L. lactis dairy isolate, lac⁺,contains 6 plasmids- (19), example 1 pLP712, pSH71, pSH72, pSH73, pSH74,herein pNZ712. 2 NCDO712 Ery^(R); harboring pIL253 with pilin operonexample 1 (pIL253pil) spaCB-spaA-srtC1-srtC2 from NCDO712 herein 3MG1363 Plasmid-cured derivative of L. lactis NCDO712 (19) 4 MG1363Ery^(R); L. lactis MG1363 harboring pIL253 with example 1 (pIL253pil)pilin operon spaCB-spaA-srtC1-srtC2 from herein NCDO712 5 MG1363Ery^(R); L. lactis MG1363 harboring pIL253 and example 1 (pIL253pilΔ1)pilin operon spaCB-spaA-srtC1-srtC2 from herein NCDO712 with 1,5-kbinternal deletion in spaCB 6 MG1299 Ery^(R); L. lactis lac⁺ derivativeof NCDO712 example 1 (pIL253pil) which additionally to pLP712 harboringthe herein pilin operon (spaCB-spaA-srtC1-srtC2) from the same NCDO712strain 7 MG1299 Derivative of L. lactis NCDO712, harbors (19) pLP712;lac⁺ 8 MG1362 Derivative of L. lactis NCDO712 (19) (described to harborpSH72) 9 MG1063 Derivative of L. lactis NCDO712 (19) (described toharbor pSH73 and pSH72) 10 MG1261 Derivative of L. lactis NCDO712 (19)(described to harbor pSH73) 11 MG1365 Derivative of L. lactis NCDO712(19) (described to harbor pSH71) 12 MG1614 Str^(R) and Rif^(R);derivative of L. lactis MG1363 (19) 13 MG1614_clu⁺ lac⁺. Transconjugantof MG1614, harbors example 1 pLP712 from NCDO712 and shows clumpingherein phenotype. 14 MG1614_clu⁻ Lac⁺. Transconjugant of MG1614, harborsexample 1 pLP712 from NCDO712 and show herein non-clumping phenotype. 15MG1363ΔacmA Derivative of L. lactis MG1363 with deletion of (55) acmA,which leads to chaining phenotype 16 MG1363ΔahrC Derivative of L. lactisMG1363 with deletion of (32) ahrC 17 IL1403ΔacmAacmD Derivative of L.lactis IL1403 with deletion of (63) acmAacmD, which leads to chainingphenotype 18 IL1403 Ery^(R); Derivative of L. lactis IL1403 harboringexample 1 (pIL253pil) pilin operon from pSH74 of NCDO712; shows hereinchaining phenotype and high hydrophobicity 19 MG1363ΔdltD Derivative ofL. lactis MG1363 with deletion of (15, 56) dltD 20 MG1363 Ery^(R);Derivative of L. lactis MG1363 harboring example 1 (pIL253) plasmidpIL253 herein 21 IL1403 Plasmid-free derivative of L. lactis IL594 (4)22 MG1363pNZ521 Cm^(R) and Ery^(R); derivative of L. lactis MG1363example 1 (pIL253pil) harboring pNZ521 and pIL253pil (spaCB- hereinspaA-srtC1-srtC2) from the NCDO712 strain 23 MG1363pNZ521 Cm^(R);derivative of L. lactis MG1363 harboring (44) proteolytic positive geneson pNZ521 24 MG1363ΔgalE Derivative of L. lactis MG1363 with deletion of(23) galE leading to chain formation without galactose in a growthmedium 25 MG1363pNZ4120 Ery^(R); derivative of L. lactis MG1363harboring (3) EPS gene cluster from B40 Plasmids 1 pIL253pil Ery^(R);13.1 kb; pIL253 harboring pSH74 pilin example 1 operonspaCB-spaA-srtC1-srtC2 with 300 bp herein upstream region 2 pIL253pilΔ1Ery^(R); 11.6 kb; pIL253 harboring spaCB-spaA- example 1 srtC1-srtC2with 1,5-kb internal deletion in herein spaCB 3 pNZ521 Cm^(R); encodesthe extracellular serine proteinase (44) (PrtP) from strain SK110 4pLP712 The 55.39 kb plasmid encoding genes for (65) lactose catabolismand a serine proteinase involved in casein degradation 5 pIL253 Ery^(R);4.9 kb; Low copy-number derivative of (49) pAMβ1 6 pNZ4120 Em^(R);pIL253 derivative containing a 17-kb (3) Ncol fragment carrying the epsgene cluster from NIZO B40

For experiments carried out in milk, full fat pasteurized andhomogenized milk (3.6 g/100 ml fat, 3.5 g/100 ml protein, 4.7 g/100 mllactose) (Friesland Campina) was purchased from a local shop andsterilized at 115° C. for 15 min to denature whey proteins and aggregatethem with caseins and fats (9). Milk was supplemented with sterile 50%glucose solution (4% final concentration) and sterile 20% Bacto™Casitone (Pancreatic digest of casein, BD, Sparks, MD, USA) solution to0.2% final concentration to ensure growth of lactose and proteasenegative strains respectively. For lactose and protease positive strainsthis supplementation was replaced by the addition of the same volume ofwater to the milk. The warm (30° C.) milk was inoculated with 2% v/vovernight culture.

Milk acidification rate and pH. Acidification rates and final pH weremeasured with a Cinac 14 ph+2T (Alliance instruments, Freppilon,France). pH electrodes were inserted into 10 ml tubes with inoculatedmilk and samples were measured for 21 h at 30° C. every 6 min.

Sedimentation profile of fermented milk. 400 μl of inoculated milk weretransferred into LUMiFuge rectangular 2 mm synthetic cells(www.lum-gmbh.de). The cuvettes were incubated statically forapproximately 21 hours at 30° C. Non-inoculated milk was used as acontrol. Sedimentation profiles of the fermented milk samples weredetermined using a LUMiFuge® 110-02 Technology (LUM GmbH, Berlin,Germany) according to the manufacturer's instructions. The rectangularsynthetic cells (2 mm, PC) with 400 μl fermented milk were centrifugedat 2325 g at 20° C. for 1 h. The measurement interval of the opticaldensity along the cuvette was 30 sec, which created 121 profiles. Thelight factor was 2. The sedimentation rates of the samples werecalculated using SepView5 to determine the slope of the integratedtransmission change in time. Slopes with the highest fit (r>0.98) wereused for sample comparison.

Matrix structure visualized with confocal laser scanning microscopy(CLSM). One ml homogenized milk was supplemented 15 μl of a solution of0.5% Acridine Orange and 0.025% Rhodamine B to stain proteins and fatdroplets. This milk was inoculated with 1% overnight culture and 1 μlSyto 9 (non-diluted solution) was directly added to the sample to stainthe bacterial cells. The inoculated milk sample was transferred to aCLSM slide with a cylindrical plastic cup attached to it which was thencovered with a lid (ref to Hassan 1995) to prevent evaporation. Thesample was incubated for 21 h at 30° C.

Images were taken using a Leica TCS SP 5 confocal laser scanningmicroscope (Leica Microsystems CMS GmbH, Mannheim, Germany) with Leicaapplication Suite Advanced Fluorescence v. 2.7.3. build 9723. The Argonlaser was used to visualize the bacteria stained with Syto9, and DPSS561 laser was used to visualize milk protein/fat droplet matrix stainedby Acridine Orange and Rhodamine B. The objective lens used was HCX PLAPO 63×/1.2/water CORR CS.

Volatile compounds with GC-MS. After the gel hardness measurements themilk samples (2.1 g) were transferred to a 10-mL dark brown glassheadspace vial and were kept at −20° C. until analyzed. After samplethawing, 50 μl of internal standard (trichloropropane 5 ppm) was addedto each sample. Volatile compounds were measured as earlier described(11) with the following changes. We used a Solid Phase Micro Extration(SPME) extraction method with a grey fiber(Carboxen/polydimethylsiloxane/divinylbenzene, Supelco, USA) for 20minutes at 40° C. The compounds were separated on a 30 m×0.25 μmFactorFour column (Agilent, The Netherlands) with a 1 ms (df=1 μm)stationary phase. The total run time, including cooling, wasapproximately 36 min Mass spectra were recorded by a DSQ massspectrometer (Thermo Fisher Scientific Inc.). The fingerprints(chromatograms) were studied to detect differences in volatile compoundsbetween the samples (control strain and derivatives). Peaks that weredifferent between samples were identified using the NIST MS Searchlibrary version 2.0. For those peaks that could be identified the peakarea was calculated using the software package Xcalibur (Thermo).

Gel strength of fermented milk. The bacterial strains were pre-culturedovernight in milk supplemented with the appropriate antibiotic ifrequired. For the gel strength and viscosity measurements, 300 ml milkwas inoculated with 2% of the strains pre-cultured as described above.The 300 ml milk samples did not contain any antibiotics to minimizedifferences between samples. The prepared milk was distributed to 100 mlsterile glass cups (70 mm diameter). The cups were incubated staticallyfor 21 hours at 30° C.

Gel strength was measured with a Texture Analyzer (TA.XTplus, StableMicro Systems Ltd., Sprundel, NL) equipped with a 5 kg load cell. 100 mlof fermented milk were compressed uniaxially to a depth of 20 mm with aconstant speed of 1 mm/sec by a probe with a grid-like geometry having10 mm side squared openings. The peak force applied on the samplecorresponds to the hardness of the milk gel.

Viscosity of fermented milk. After the texture analysis, the viscosityof the fermented milk was measured with a rotational viscometer (HaakeSearle RV20 Rotovisco and RC 20 Rheocontroller, ThermoScientific,Hofheim, Germany) with MV2P (middle viscous profiled) rotor. 60 gfermented milk was transferred to the MVP cup and allowed to rest for 15min After resting the sample was measured at the shear rate graduallyincreased from 0 to 400 s⁻¹ for 5 min and then decreased from 400 to 0s⁻¹ for 5 min

Results

Surface Altered Lactococci

To investigate if changes in cell surface properties affect productfunctionality, we performed assays using isogenic Lactococcus lactisstrains that differed in surface charge, hydrophobicity, chaining,clumping, attachment to proteins, pili expression and EPS production(Table 5).

TABLE 5 Phenotypic characteristics of L. lactis strains (n = 3) in astationary growth phase at pH of 6.7. Charge has negative values and isshown in mV, CSH stands for cell surface hydrophobicity, Att_NaCN - forattachment to sodium caseinate, Att_NaNC90C - for attachment to sodiumcaseinate heated at 90° C. for 10 min, Att_ParaCN - for attachment toparacaseinate, and E24 means emulsion stability after 24 h. Att_NaCAtt_NaCN90 Att_ParaC L. lactis ZP, mV CSH, % N, % C, % N, % E24, % Pilioverexpressing strains are chaining and clumping IL1403  −9.2 ± 0.5    0 ± 19.89 41.65 ± 13.59 67.22 ± 2.24  63.66 ± 1.56  0 ± 0IL1403(pIL253pil) −13.87 ± 1.25 93.24 ± 4.47 1.79 ± 0.66 0 ± 0 29.36 ±15.34 88.89 ± 19.25 MG1299 −30.25 ± 2.05 75.48 ± 3.26 82.83 ± 2.7  82.71 ± 0.27  79.23 ± 2.16   35.6 ± 24.85 MG1299(pIL253pil)  −18.1 ±1.29 99.06 ± 1.16  27.9 ± 24.22 0 ± 0 30.22 ± 52.34 99.33 ± 0.58 MG1363pIL253 −26.4 ± 1.8  3.75 ± 6.84 17.7 ± 8.65 37.86 ± 25.39 80.56 ±6.19  0 ± 0 MG1363(pIL253pil) −11.87 ± 0.21 94.03 ± 2.16 4.61 ± 2.931.88 ± 0.59  6.12 ± 10.59 85.24 ± 15.01 MG1363pNZ521 −31.67 ± 0.76 18.74± 10.9 90.34 ± 2.76  88.82 ± 2.43  79.17 ± 8.96  0 ± 0 MG1363pNZ521−16.97 ± 1.89   99 ± 1.04 0 ± 0 0 ± 0 0 ± 0 65.56 ± 29.88 (pIL253pil)NCDO712  −19.7 ± 0.46 99.44 ± 0.32 96.02 ± 0.64  95.68 ± 2    94.74 ±0.6   99.67 ± 0.58  NCDO712(pIL253pil) −20.5 ± 1.3 96.31 ± 0.62 1.74 ±0.5  37.95 ± 9.96  35.08 ± 16.81 100 ± 0   Chaining phenotypeIL1403ΔacmAacmD −12.03 ± 0.35  9.48 ± 4.58 97.17 ± 0.6   96.24 ± 2.07 96.37 ± 1.27  0 ± 0 MG1363ΔacmA  −31.4 ± 1.42 15.53 ± 4.48 88.95 ± 0.68 87.56 ± 0.97  83.51 ± 9.96  0 ± 0 MG1363ΔgalE −28.73 ± 0.76 14.91 ± 4.4494.24 ± 2.05  95.63 ± 0.53  37.49 ± 24.57 0 ± 0 MG1363ΔdltD −29.17 ±0.21 15.38 ± 3.52 84.94 ± 0.62  83.91 ± 3.05  82.4 ± 3.16 0 ± 0Non-chaining, non-clumping phenotype MG1363  −30.2 ± 0.66 5.78 ± 0.282.3 ± 0.9  82.77 ± 1.95  79.38 ± 1.73  0 ± 0 MG1261 −30.03 ± 1.16 21.76± 0.2  93.96 ± 1.39  92.55 ± 0.83  88.76 ± 6.29  0 ± 0 MG1063    −29 ±0.87 16.67 ± 0   89.47 ± 2.64  89.61 ± 2.05    83 ± 5.07 0 ± 0 MG1362−31.67 ± 3.25  5.56 ± 9.62 89.58 ± 3.43  84.93 ± 9.73  79.62 ± 14.1  0 ±0 MG1365 −30.33 ± 1.16 28.59 ± 6.12 92.92 ± 1.76  93.1 ± 1.27  76.6 ±26.36 0 ± 0 MG1614_clu⁻ −39.4 ± 0.5 73.85 ± 4.05 96.34 ± 0.88  94.64 ±0.63  89.4 ± 4.95 0 ± 0 MG1363ΔahrC −29.53 ± 1.21  79.93 ± 10.68 84.47 ±1.12  81.01 ± 2.73  80.24 ± 1.68  23.81 ± 2.06  MG1363 −16.93 ± 0.87 79.67 ± 16.92   66 ± 10.93 64.53 ± 14.39 86.28 ± 5.22  97.78 ± 3.85 (pIL253pilΔ1) Clumping phenotype MG1614 −41.97 ± 2.43 20.39 ± 3.12 97.12± 0.8   94.83 ± 4.11  97.66 ± 1.25  0 ± 0 MG1614_clu⁺ −35.97 ± 0.4 90.18 ± 3.74 81.69 ± 15   58.13 ± 26.29 94.6 ± 0.64 31.33 ± 4.04  EPSproducing MG1363  −30.2 ± 0.66 5.78 ± 0.2 82.3 ± 0.9  82.77 ± 1.95 79.38 ± 1.73  0 ± 0 MG1363pNZ4120  −18.8 ± 2.19    0 ± 23.76 96.17 ±1    98.27 ± 0.52  97.37 ± 0.15  0 ± 0

The 25 strains included variants with different plasmids from the dairyisolate NCDO712 and strains in which particular genes were deleted orover-expressed. Deletion of acmA, acmAacmD, dltD and galE lead to a cellchaining and introduction of pNZ4120, a plasmid encoding the EPS genecluster from L. lactis NIZO B40, lead to EPS production in MG1363 and anincreased surface charge. We recently isolated transconjugant strains ofMG1614 obtained by conjugation of the plasmid pLP712 from NCDO712.(Tarazanova et al. manuscript in preparation) (4). All transconjugantsisolated during these experiments were indeed shown to carry theprotease/lactose plasmid pLP712 (12). However, although there wastransfer of pLP712 other changes occurred as two other phenotypes wefound one which gives loose cocci and has a high surface hydrophobicity(designated MG1614_clu⁻), and the other phenotype which showed aclumping phenotype and the same high hydrophobicity (designatedMG1614_clu⁺) (Tarazanova et al. manuscript in preparation). Other MG1363derivatives such as MG1365, MG1362, MG1063, MG1261, MG1299 differ fromMG1363 by carrying of 1 or 2 plasmids from NCDO712 (shown in Table 4).Recently identified is a lactococcal gene cluster encoding thespaCB-spaA-srtC1-srtC2 genes whose overexpression results in theappearance of pili like structures on the cell surface (example 1). Thispili expression results in chain formation, cell clumping and a highsurface hydrophobicity. A strain with an internal deletion of 1.5 kb inspaCB (designated as MG1363(pIL253pilΔ1)) does not show chaining andclumping but it retains high cell surface hydrophobicity. In this strainpili seem to be expressed but are not attached to the cell surface.These strains have also been used in this analysis (Table 5).

Acidification Rates

As the acidification rate and the final pH during milk fermentation caninfluence textural properties we followed the pH for all strains during21 h of milk fermentation (Table 6). Fast acidification of milk resultsin excessive syneresis, while very slow milk acidification leads to aformation of a weaker gel (21, 33, 36-39). While the maximumacidification rates for most strains were ˜0.5 pH/h, strains showing achaining phenotype (MG1363ΔacmA, MG1363ΔgalE) the pilin harboring strainMG1363(pIL253pil) and its control strain MG1363(pIL253) had slowermaximum acidification rates (Table 6). The final pH for all milk samplesfermented by derivatives of L. lactis MG1363 strains was 4.25±0.04, withthe exception of the proteolytic positive strain harboring piliMG1363pNZ521(pIL253pil) and strain IL1403 and derivatives.

TABLE 6 End pH and maximum acidification rate for selected L. lactisstrains Max acidification L. lactis strain pH rate (pH/h) NCDO712 4.24 ±0.06 −0.51 MG1363 4.24 ± 0.05 −0.53 MG1362  4.2 ± 0.04 −0.52 MG1063 4.19± 0.05 −0.53 MG1261 4.21 ± 0.05 −0.54 MG1365  4.2 ± 0.05 −0.50MG1363(pIL253) 4.29 ± 0.05 −0.36 MG1363(pIL253pil) 4.23 ± 0.05 −0.39MG1299 4.23 ± 0.05 −0.49 MG1299(pIL253pil) 4.27 ± 0.05 −0.54 MG1363ΔacmA4.24 ± 0.05 −0.42 MG1363ΔahrC 4.28 ± 0.03 −0.56 MG1363ΔdltD 4.24 ± 0.05−0.52 MG1363ΔgalE 4.21 ± 0.05 −0.45 MG1614 4.23 ± 0.05 −0.54MG1614(pLP712)_cl⁺ 4.27 ± 0.05 −0.50 MG1614(pLP712)_clu⁻ 4.27 ± 0.06−0.50 MG1363pNZ4120 4.34 ± 0.07 −0.53 MG1363pNZ521(pIL253pil) 4.77 ±0.04 −0.46 MG1363pNZ521 4.22 ± 0.05 −0.49 IL1403 4.45 ± 0.02 −0.53IL1403ΔacmAacmD 4.39 ± 0.01 −0.51 IL1403(pIL253pil) 4.47 ± 0.01 −0.51

Sedimentation profiles (LUMiFuge). To investigate the influence of cellsurface properties on milk stability, sedimentation profiles weremeasured. The principle of the measurement is that transmission of thesample is measured over the complete height of a cuvette in time duringcentrifugation of the cuvette. If a milk sample is stable, particlesadsorb the light, and transmission remains low over the complete samplesize. If the sample is not stable during centrifuging, sedimentationfrom the top to the bottom of cuvette is detected through an increase intransmission on the cleared part of the sample. Our results indicate alow sedimentation slope for milk fermented with the EPS producingMG1363pNZ4120 which is consistent with the fact that EPS binds watermolecules, and therefore, exhibits lower syneresis and high viscosity(2, 3, 5, 8). We found that neither the over-expression of pili onsurface of lactococcal bacteria nor cell chaining or clumping did affectthe sedimentation profile when compared to control strains.

Localization of cells in fermented milk. To investigate if alterationsof the bacterial cell surface affect the location of cells in fermentedmilk we used confocal laser scanning microscopy (CLSM). Images fromundisturbed coagulated milk were taken from samples that were fermentedon the imaging slide itself. Due to the sedimentation of cells duringthe fermentation and the limited depth at which CSLM allows imaging (˜40μm) the number of cells in the image might be higher than in the middleof a sample. However, the visualized effect of surface properties on thebacterial location in a fermented dairy product is indicative for whatoccurs throughout the product. Imaging of the undisturbed fermented milktherefore provides information of the behavior of the bacteria in situ,as the components and the undisturbed structure of the fermented productcan be observed. The results showed obvious differences between variousstrains. For example loose cocci and clumping cocci locate in theprotein matrix close to serum regions (FIGS. 7, A and C); aggregatedcells of MG1363(pIL253pil) tightly fill the cavities between thecoagulated proteins in fermented milk (FIG. 7, B); chaining cellslocalize in a freeway in milk matrix: go through proteins and serumregions as well as locate in close proximity to those regions (FIGS. 7,D and E); EPS producing cells locate in serum regions (FIG. 7, F).Interestingly, the localization effect of pilin over-expression inMG1363 was not observed in strain IL1403 indicating that such effectmight be background dependent. Overall CLSM results indicate that thesurface properties can have a profound effect on the bacterial locationin the food matrix.

Flavour volatiles. To determine the effect of altered surface propertieson flavour volatiles of fermented milk we analyzed the volatilecompounds in the headspace of fermented milk using GC-MS. The analysisresulted in some statistically significant changes in flavour volatiles.For example, pili over-expression led to chaining and clumping andstatistically significantly increased the formation of flavour volatilesduring milk fermentation. This phenomenon might be explained by highlyincreased (>90%) hydrophobicity, and the bacteria with more hydrophobicsurfaces have a bigger affinity for aroma compounds (7). However,because the magnitude of the differences is limited, less than 10 fold,all significant differences in flavour can probably not be perceived.This means that with the present invention the texture of fermented milkcan be changed without changing the flavour.

Gel hardness. We measured gel hardness and viscosity to determine ifcell surface properties influence the rheological parameters offermented milk. The gel hardness for MG1363(pIL253pil) increased byapproximately 46% compared to control MG1363(pIL253) (p=0.009). Asimilar trend was observed for a proteolytic-positive strainoverexpressing pili (MG1363pNZ521(pIL253pil) which increased the milkgel strength by 15% (p=0.04). For other three strainsNCDO712(pIL253pil), MG1299(pIL253pil), and IL1403(pIL253pil) the gelhardness did not change compared to their control strains (p>0.05). Theresults for IL1403(pIL253pil) could be explained by very low amount ofpili on the surface of this bacteria compared to MG1363(pIL253pil). Wewould like to note that the acidification rates were similar for thesamples MG1363(pIL253) and MG1363(pIL263pil) and for proteolyticpositive samples MG1363pNZ521 and MG1363pNZ521(pIL263pil), and can,therefore, be excluded as influencing factor on gel hardness. We alsoinvestigated if the combination of pili producing strains with EPSproducers in the same fermentation leads to synergy effects on gelhardness, but did not find such effects (Table 7).

Gel hardness measurements showed that it is significantly altered byusing cells that either form chains or have a clumping phenotype.IL1403AacmAacmD, a chain forming derivative of IL1403, decreased the gelhardness when compared to its control strain IL1403. A similar trend wasobserved for chaining MG1363AacmA when it is compared to its controlMG1363. The clumping strain MG1614_clu⁺ increased gel hardness by 4.3%compared to its control MG1614, however, changes in gel hardness below10% are thought to not be perceived in the mouth. As an increase in gelhardness is not seen for all strains where the pili were overexpressedwe conclude that the effect is strain dependent which can be explainedby different surface properties of the strains used. Together theseresults show that bacteria with pili on its surface can significantlyenhance the milk gel strength.

TABLE 7 Gel hardness (g) and viscosity (mPa · s) of milk fermented bysurface altered lactococci. The results show the average of 3 biologicalreplications. Gel Viscosity, Strain hardness (g) pH mPa · s Pilioverexpressing strains compare to their control strains without piliMG1363(pIL253) 40.1 ± 2.18 4.29 ± 0.05 722.7 ± 11.3  MG1363(pIL253pilΔ1) 45.1 ± 1.13* 4.23 ± 0     1050 ± 19.1* MG1363(pIL253pil) 47.8 ± 1.8*4.23 ± 0.05 1079.3 ± 58.2*  MG1363pNZ521  42.8 ± 0.4   4.22 ± 0.05  773± 64.3 MG1363pNZ521(pIL253pil) 49.1 ± 2.8*  4.34 ± 0.006 1027.2 ± 30*   NCDO712 43.5 ± 0.6  4.24 ± 0.06  1498 ± 60.6  NCDO712(pIL253pil) 42.4 ±3.2   4.23 ± 0.006   1192 ± 146.2* MG1299 38.59 ± 0.6   4.23 ± 0.05726.1 ± 68.1  MG1299(pIL253pil) 38.2 ± 0.6   4.27 ± 0.001 797 ± 54 IL1403 37.5 ± 0.64 4.45 ± 0.02 680.1 ± 34.5  IL1403(pIL253pil)  37 ± 0.7 4.47 ± 0.001 745 ± 78  Combinations of EPS and pili/no-pili formingstrains MG1363pNZ4120 48.9 ± 1.9   4.32 ± 0.006  4556 ± 37.7*MG1363pNZ4120+MG1363(pIL253pil) 51.9 ± 1.1   4.22 ± 0.006  3231 ± 78.9 MG1363pNZ4120+MG1363(pIL253pilΔ1) 44.4 ± 3.6   4.19 ± 0.006  2921 ±277.7 MG1363pNZ4120+MG1363pIL253 46.9 ± 2.9   4.24 ± 0.006 3242.7 ±396.7  MG1363pNZ4120+NCDO712 45.6 ± 6.8  4.18 ± 0.01  2847 ± 117.1MG1363pNZ4120+NCDO712(pIL253pil) 46.97 ± 1.48  4.21 ± 0     2976 ± 105.5Chaining strains IL1403  37.5 ± 0.64  4.45 ± 0.02 680.1 ± 34.5 IL1403ΔacmAacmD  35.4 ± 0.14* 4.39 ± 0.01 1008.5 ± 109.3* MG1363 47.2 ±2.08 4.24 ± 0.06 926.1 ± 50.5  MG1363ΔacmA  41.5 ± 1.08* 4.24 ± 0.051034.7 ± 18.9*  Transconjugants (clumping/non-clumping) MG1614  40 ±0.43 4.23 ± 0.05 832.5 ± 40.9  MG1614_clu⁻  39 ± 1.19 4.27 ± 0.06 739 ±37* MG1614_clu⁺ 41.7 ± 1   4.27 ± 0.05 887.9 ± 28    NCDO712 derivativescarrying 1 or 2 plasmids; MG1363 derivatives with deletion of 1 geneNCDO712 42.4 ± 1.3   4.24 ± 0.06 867.2 ± 106.1 MG1363 43.78 ± 0.89  4.24 ± 0.05 926.1 ± 50.5  MG1363ΔahrG 43.8 ± 0.43  4.28 ± 0.03   771 ±205.5 MG1363ΔdltD 43.9 ± 0.56  4.24 ± 0.05 891.9 ± 66.9  MG1363ΔgalE44.71 ± 0.58   4.21 ± 0.05 871.1 ± 181.3 MG1063 45.17 ± 0.39   4.19 ±0.05 986.97 ± 20.7   MG1261  41 ± 0.91 4.21 ± 0.05 790.97 ± 20.6  MG1362 44.1 ± 0.81   4.2 ± 0.04 945.1 ± 40.9  MG1299 38.59 ± 0.6    4.23± 0.05 726.1 ± 68.1  *Significant at p < 0.05-comparisons were made tonon-surface modified controls with the same gray-color code in the rowsabove.

Viscosity was increased by 19-35% in milk fermented withMG1363(pIL253pil) (p=0.0005) and MG1363pNZ521(pIL253pil) (p=0.003)compared to the control conditions fermented with MG1363pIL253 andMG1363pNZ521, respectively (Table 7). An increased viscosity, howevernot significant, was observed for pili overexpressing strains such aslactose/protease positive strain MG1299(pIL253pil) (p=0.23) and for L.lactis IL1403(pIL253pil) (p=0.26). In comparison to milk fermented withthe EPS producing strain MG1363pNZ4120 the increase in viscosity causedby pili production is lower. There was no synergy effects found whenmilk was fermented with a 1:1 mixture of EPS and pili producing strains.However it is likely that that an up to 35% increase in viscosity causedby pili expression is a substantial alteration of food texture. Whencells produced a high number of pili they also showed increasedchaining. These strains formed milk gels with increased viscosity. Forexample, the chaining strain MG1363AacmA and IL1403AacmAacmD formed milkgel with a viscosity which increased by 11% and by approximately 50%compared to their controls MG1363 (p=0.025) and IL1403 (p=0.026) shownin Table 7. Acidification rate can be excluded as influencing factor onmilk viscosity for it is similar for the compared samples. The resultsof milk viscosity show that surface properties such as cell aggregation,chaining, formation of EPS and pili, increase product thickness in therange of 20-48%.

Correlation Between Surface Properties and Textural Parameters ofFermented Milk Product.

No correlation was seen between cell surface charge, surfacehydrophobicity, attachment to proteins, and emulsion stability, theseare all independent parameters. However, the surface morphology such asclumping, expression of pili, and EPS formation influence the charge,hydrophobicity and attachment to proteins. For example, by makingcorrelations for only pili-overexpressed stains and its control strainswe saw that the presence of pili on bacterial surface led to increasedsurface hydrophobicity and, consequently, emulsion stability (r=0.91),while in general emulsion stability and hydrophobicity are independentparameters. Furthermore, clumping, over-expression of pili and EPSformation influence the gel hardness and viscosity (r>0.82).Importantly, no correlation was seen between pH and viscosity (r=−0.07)and between pH and gel hardness (r=−0.59). Thus, chaining, clumping,formation of pili or EPS influence both the cells surface properties andtextural parameters of a product.

Discussion.

Here we investigated the potential role of bacterial surface propertiesand morphologies on the production of volatile metabolites, thestructure, and the textural parameters of fermented milk. The surfaceproperties of lactococci were modified in terms of chaining, clumping,exopolysaccharide formation, and pili over-expression. All surfacealterations were in an isogenic background to be able to compare theresults. Furthermore, the milk composition, milk pre-treatment andfermentation conditions were the same to eliminate the influence ofthese factors on the results. The acidification rate of used strains wasalso measured to be sure that the acidification speed between strain ofinterest and its control strains are similar. Additionally, pH offermented milk did not correlate with milk viscosity and gel hardness.Therefore, all these mentioned parameters can be excluded as influencingfactors on structure, texture, and volatiles' formation of fermentedmilk. The results not only demonstrate that alteration of cell surfaceproperties (chaining, clumping, formation of exopolysaccharides, piliover-expression) affect cell surface charge, hydrophobicity and theattachment to proteins, but also textural parameters of fermented milk,including viscosity, gel hardness and the bacterial localization in thefood matrix (Table 8). Based on the observed effects of cell surfacecomposition on textural parameters of fermented milk, we hypothesizethat bacteria could be considered as structure element in a food matrix.

TABLE 8 The summary of textural parameters of milk gel fermented withsurface altered lactococci. Phenotype Strain Viscosity Gel hardness Piliover- MG1363(pIL253pil) ↑* ↑* expression MG1363(pIL253pilΔ1) ↑* ↑*MG1363pNZ521(pIL253pil) ↑* ↑* MG1299(pIL253pil) ↑ ↑ NCDO712(pIL253pil) ≈↓* IL1403(pIL253pil) ↑ ≈ Chaining IL1403ΔacmAacmD ↑* ↓* MG1363ΔacmA ↑*↓* Clumping Transconjugant 5 ↑ ↑* ↑ - increased; ≈ - did not change; ↓ -decreased; * - significance (p < 0.05)

As structure element bacterial cells possess amphiphilic surfaceproperties, so the cells are charged and at the same time havehydrophobic areas which are determined by the complex molecularcomposition of cell wall. For example, the pili-overexpression in thestrain MG1363pIL253 led in neutral pH to decreased charge from −26.4±1.8mV to −11.87±0.21 mV, and increased hydrophobicity from 5% to 96%. Thecharge decrease can be explained by the positive net-charge of pilinproteins. Additionally, the charged residues of the pilin protein shouldbe reflected on the outside of the pilus because charged residues in thecore of a tight 3D structure are generally unfavorable; and mostpositive charge is from arginine which is quite bulky and doubleoccurrences of a few positively charged amino acids would hinder proteinfolding even further (66). However, the surface of lactococcal bacteriais much more complex; therefore, the overall net surface charge of piliexpressing strains remains negative. As well as having positively andnegatively charged regions, pili have 30-40% hydrophobic patches (basedon protein sequence analysis) which would be buried in the proteinmolecule or they will be involved in the inter-protein connectionsduring pili organization. Possibly these pili hydrophobic patches areresponsible for the increase in the cell surface hydrophobicity from5-20% to 99%.

Bacterial surfaces engage in physico-chemical interactions with milkcomponents. The cell surface composition is the polymeric layer. Thereare various interactions that can occur between the molecules of thepolymeric layer of bacteria and milk components or between cell surfacesof two bacteria: Van der Waals interactions (29), electrostaticrepulsions and attractions, hydrogen bonds, hydrophobic interactions,salt bridges and steric interactions (6, 60) (FIG. 9). The combinationof all of these interactions at the same time is also possible. Thestructure, stability and textural properties of fermented milk matrixare determined by the type of microbe-matrix interaction and interactionforce. The type of microbe-matrix interactions depend not only on thepH, the bacterial localization in milk before fermentation, the nutrientdistribution in milk matrix, the roughness of the cell surface but alsoon the strain itself. Importantly, for most lactococcal cells, pH doesnot influence the cell surface charge, it constantly remains negative.Modifying the cell surface morphology, changes are seen surfaceproperties, and consequently microbe-matrix interactions. The end resultof this all is that the structure and textural parameters of milksignificantly change.

Possessing amphiphilic properties and having complex surface molecularcomposition, as a structural element bacteria can have several roles ina fermented milk: 1) interact with proteins and other milk components ornot, 2) can locate in serum regions or in protein matrix, and 3) formclumps or not. Based on these three items, the impact of bacteria ontextural milk properties can be found. Considering the first item,bacterial surfaces engage in physico-chemical interactions with milkcomponents. The cell surface composition we can call polymeric layer.Between the molecules of the polymeric layer of bacteria and milkproteins or between cell surfaces of two bacteria combination of severalinteractions can occur at the same time: Van der Waals interactions(26), electrostatic repulsions and attractions, hydrogen bonds,hydrophobic interactions, salt bridges, and steric interactions (5, 57).For instance, for LGG the force between micellar casein (or denaturedwhey) protein and cell is about 0.4 nN (6), and it is verystrain-dependent. The type of microbe-matrix interaction and forcestrength between microbe-matrix seem to determine the structure,stability, and textural properties of fermented milk matrix. The type ofmicrobe-matrix interactions depend on the pH, the bacterial localizationin milk before fermentation, the nutrient distribution in milk matrix,the roughness of the cell surface and the strain itself. Importantly,for most lactococcal cells, the pH does not influence the cell surfacecharge, it constantly remains negative. We saw that by modifying thecell surface morphology, we change surface properties, and consequentlymicrobe-matrix interactions. As a result, the structure and texturalparameters of milk significantly change. In milk (pH 6.7) electrostaticrepulsion occurs between negatively charged casein molecules and betweenmilk proteins and negatively charged bacterial cells through presence ofacid/base groups on their surfaces (e.g. in proteins). Electrostaticinteractions depend on the pH and ion concentration in milk. At the sametime the cell surface contains also hydrophobic patches (surfaceproteins, pili) which interact with hydrophobic patches of milk proteinsor other bacterial surface molecules (pili or other surface proteins).Hydrophobic interactions are the strongest and its strength increaseswhen the temperature increases (59). During milk fermentation the pHdrops from 6.7 to 4.2 via coagulation point 4.6 of caseins (25). Proteinaggregation starts already at pH 5.2-5.3, and at pH 4.6 casein micellesaggregate, because the negative charge of caseins is shielded by H⁺ oflactic acid produced by the bacteria. Subsequently, hydrophobic patchesof casein molecules unfold and milk proteins start to aggregate viahydrophobic interactions. At this point bacterial cells which do notinteract with milk components become incorporated in the milk gelnetwork. When the pH decreases below coagulation pH 4.6 of caseinmicelles, milk proteins become positively charged. In this case thebacterial attachment to milk proteins is governed by a combination ofinteractions including electrostatic and hydrophobic forces.

The second and third mentioned roles of bacteria were cell location inserum regions or in protein matrix and formation of chains/clumps whichincreases fermented milk viscosity and gel hardness. The microscopicobservations of the cell localization of pili over-expressing cells inthe milk matrix seem to show a more dense milk gel structure. Thesecells seem to be localized in the serum regions (FIG. 7 and FIG. 8,panel B). Pili over-expression increased the viscosity of fermented milkup to 35% (Table 7). This was seen independently for three piliover-expressing strains: MG1363(pIL253pil), MG1363pNZ521(pIL253pil),MG1299(pIL253pil), but not for NCDO712(pIL253pil) and IL1403(pIL253pil),which indicates that this effect might be background dependent. NCDO712already harbors the pilin genes which make differences upon increasingthe expression less pronounced. For IL1403 electron microscopy showedrelatively few pili on the surface which might explain our findings.Importantly, a 20% increase in viscosity is likely to be perceived in amouth and it would be interesting to test this in a sensory analysis.Increased viscosity might be explained by the increased solid content ofproteins aggregates with cell aggregates in the milk serum phase. Herethe size and shape of particles is of importance: cells form very strongaggregates and locate in serum cavities which mean the amount of freewater is decreased.

As it was mentioned above, chaining bacteria (acmAlacmD deletions)increased viscosity up to 12-48%, but decrease gel hardness by 6-14%(Table 7, FIG. 8, panel C). The decreased gel hardness might beexplained by cavities of serum in milk matrix: they do not provide anyadditional bonds to make gel stronger, and also possibly that the cells,which do not interact with the aggregated caseine micelles, are thus notincluded in the matrix but end up more like structure breakers, it meansthe matrix has to form around them. The increased viscosity of milkmatrix fermented with chaining cells might be explained by cellmorphology, cell size e.g. length is of importance here. No changes involatile formation were detected for chaining strains compared tonon-chaining controls. It could be explained by unchanged cell surfacehydrophobicity leading to inability to bind more volatile molecules orsimply because chaining does not affect the formation of any volatiles.

Such cell localization in the cavities of protein matrix, cell surfaceroughness, less cavities in the protein matrix, cell chaining, strongforces between cell-cell connections, attraction forces betweencells-proteins, all these parameters seem to provide high viscosity andgel strength to milk matrix. Overall our findings suggest that byaltering surface properties of dairy starter cultures it is possible tochange product properties such as gel hardness and milk viscositywithout changing the volatile profile. It will be highly interesting tosee if the indicated properties can be perceived in sensory analysis.This would open possibilities to develop new concepts to improvefermented products.

Example 3. Bacteria as Structural Elements in a Fermented Food Product;Cell Distribution in Whey and Curd

A functional property investigated in cheese manufacturing was ifalterations on the cell surface of a dairy bacterial strain according tothe invention changes the fraction of cells that remain in curd duringcheese making. The results are listed in Table 9 here below. In contrastto earlier believes that in general roughly 90% of the cells remains inthe curd and 10% is in the whey, the data showed variations from ˜50% to˜80% of cells remaining in the curd with a bacterial cell that is not abacterial cell according to the invention. Introduction of the emptyvector pIL253 into strain MG1363 increased the fraction of cells in thecurd from ˜50% to ˜86%. This is intriguing as the empty vector harboursonly replication genes and an antibiotic marker. However, we did seewith electron microscopy that the introduction of the empty pIL253plasmid leads to an altered cell morphology. When overexpressing thepilin operon from pIL253 the fraction of cells remaining in curdincreases further to ˜98%. In cheese produced with an EPS producingstrain only (MG1363pNZ4120) ˜50% of cells remained in the curd while thecombination of this strains with pili producing strains duringmanufacturing increased the cell fraction in curd to 99%. Together thisindicates significant effects of surface properties/decoration on thecell distribution in curd and whey during cheese making using a dairybacterial strain according to the invention.

TABLE 9 Cell distribution in whey and curd (%) after cell surfacealterations. Fraction in curd CFU/ml whey CFU/g curd (%) STRAIN MEAN SDMEAN SD MEAN SD Pili overexpression MG1299 4.13E+07 2.33E+07 3.74E+083.19E+08 79.30 16.62 MG1299pil 7.05E+05 1.19E+05 4.02E+08 2.46E+07 99.82 0.04 IL1403 4.23E+06 3.46E+06 1.03E+07 6.66E+06 65.43 31.23 IL1403pil2.72E+05 1.17E+04 5.98E+07 9.28E+06 99.54  0.09 MG1363 1.54E+07 1.28E+071.42E+07 1.13E+07 49.73  8.24 MG1363pIL253 4.69E+06 2.98E+06 3.06E+071.56E+07 86.75  4.19 MG1363pil_28 1.76E+06 1.43E+06 8.08E+07 7.29E+0797.41  0.82 MG1363pil_17 6.62E+05 3.48E+05 1.73E+07 1.18E+07 95.71  1.25Mixture of EPS and pili producing strains MG1363pNZ4120 5.62E+061.18E+06 6.77E+06 1.99E+06 54.13  5.17 MG1363pi1 + MG1363eps 5.23E+053.73E+05 4.48E+07 3.45E+07 98.75  0.29

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1. A method for gene transfer by conjugation comprising providing acomposition comprising at least two bacterial strains, wherein at leastone bacterial strain comprises an increased amount of expression productof a pilin gene cluster, wherein the amount of expression product isincreased in view of Lactococcus lactis subsp cremoris NCDO712, andincubating the composition under conditions conducive to conjugation. 2.The method according to claim 1, wherein the pilin gene cluster has anucleotide sequence that has at least 30% percentage sequence identitywith SEQ ID NO: 1, or a part thereof.
 3. A method for the production ofa desired bacterial strain comprising a genetic trait of interest,comprising a) providing a composition comprising: a precursor of thedesired bacterial strain, wherein the precursor lacks the genetic traitof interest, and a donor bacterial strain comprising the genetic traitof interest, wherein at least on bacterial strain in the compositioncomprises a pilin gene cluster, b) incubating the composition underconditions conducive to conjugation, and c) optionally isolating thedesired bacterial strain comprising the desired genetic trait ofinterest.
 4. The method according to claim 3, wherein the pilin genecluster has a nucleotide sequence that has at least 30% percentagesequence identity with SEQ ID NO:
 1. 5. The method according to claim 3,comprising culturing the precursor under conditions that are conduciveto the development of clumping and/or chaining properties.
 6. The methodaccording to claim 3, wherein the bacterial strain comprising thedesired genetic trait of interest is a dairy bacterial strain.
 7. Themethod according to claim 6, wherein the dairy bacterial strain is astrain with clumping and/or chaining properties.
 8. The method accordingto claim 7, wherein the clumping property results in clumps of at least20 bacteria per average clump and wherein the chaining property resultsin chains of at least 8 bacteria per average chain.
 9. The methodaccording to claim 6, wherein the dairy bacterial strain is a lacticacid bacterial strain.
 10. The method according to claim 7, wherein thedairy bacterial strain is a lactic acid bacterial strain.
 11. The methodaccording to claim 10, wherein the lactic acid bacteria is selected fromthe group consisting of Lactobacillus, Lactococcus, Leuconostoc,Carnobacterium, Streptococcus, Bifidobacterium, Bacteroides,Eubacterium, Clostridium, Fusobacterium, Propionibacterium,Enterococcus, Staphylococcus, Peptostreptococcus, and Escherichia. 12.The method according to claim 11, wherein the lactic acid bacteria isLactobacillus.